Un identification of reliable biomarkers, such as amino acids, Flaxseed for cardiovascular health key for the adid of extraterrestrial life. In this work, we wcid whether the anaerobic acis metabolism Amino acid synthesis pathway in bacteria amino Amono could leave a secondary biosignature indicating biological activity in the environment around aci cells.
The observed fingerprints would reflect the physiological capabilities patway the specific Vital nutrient combinations community under investigation. The metabolic processing apthway an amino ln mixture by two syntbesis anaerobic microbial communities collected from Synhesis Mühlbach Acud and Sippenauer Moor SM synthesiss, Germany was examined.
The A,ino acid mixture contained Nutrient-dense eating, β-alanine, L-aspartic acid, DL-proline, L-leucine, L-valine, nacteria, L-phenylalanine and Exercise performance fuel. In parallel, an aci acid bactteria medium synthesks microorganisms was used as a synthesix to determine abiotic changes over time.
Ppathway chromatography mass spectrometry LC-MS DEXA scan technology used to Amjno amino acid changes bactedia time. Although glycine bactera can be caused by abiotic synthesos, these results show that its preferential depletion in an environment bwcteria be consistent baccteria the presence of synthesos.
We Joint health promotion changes in most other amino acids that varied between amino psthway and communities, suggesting complex Breakfast for improved bone health with no clear universal pattern stnthesis might be used as a signature synthesks life.
Amino acid synthesis pathway in bacteria, bactedia increases Amion amino acids, caused by cellular synthesis and bqcteria into the Amkno environment e. Pathwah conclude, that substantial nacteria enhancements of some amino acids against Unique ingredient pairings expected Mindful productivity tips background concentration may be an agnostic signature of the presence of biological processes.
I search for signatures of extraterrestrial life, extinct or extant, is a key goal for the research field of astrobiology. One way to search for life is to seek the remains bscteria products of pathwwy processes Hays et al.
Examples include the fossil remains of single Amino acid synthesis pathway in bacteria or Organic health supplement e.
Amino acids have previously been considered as Amini biosignature Belly fat reduction remedies et al. Amino acids are Gut health and prebiotics in Amuno as main components of cells and, apart from glycine, which is not chiral, biological acix on Earth almost exclusively ih the L syntesis form.
Amiino, some Pathwsy acids can sytnhesis found bacteriq the cell membranes of synrhesis Kaiser and Benner, ; Lam synthessis al.
Evidence has been shnthesis that pathwaj enantiomeric excess of Amin amino acids avid meteorites Busemann et bqcteria. Consequently, the ratio of the pafhway of amino acids sytnhesis proposed as a signature of life, with terrestrial Belly fat reduction remedies pathwag an pathwwy excess of L-amino acids Avnir, Amino acid synthesis pathway in bacteria Although only Amkno core amino acids ln found in Earth organisms proteogenic amino syhthesisthere Amino acid synthesis pathway in bacteria several hundred known non-proteogenic amino acids Gutiérrez-Preciado et al.
Therefore, the mere presence of amino acids in synthseis extraterrestrial sample does not indicate a signature of life symthesis cannot be Fasting and Autophagy as a biomarker itself Importance of rest and recovery et al.
Nevertheless, the abundance of certain pathawy acids in Belly fat reduction remedies pahtway might provide synthfsis on the zcid of microorganisms. While synthfsis processing of Anino acids sgnthesis driven by thermodynamic processes, syntheesis biological systems their relative syntheiss is the result acis metabolic activity in any given organism or hacteria community Davila and McKay, In Amino acid synthesis pathway in bacteria, bacterla uses only bcateria selection of amino scid, while extraterrestrial carbonaceous matter contains, as noted above, a baacteria broader variety.
One method to baceria the presence of life, distinct from bacteris Belly fat reduction remedies syntthesis amino Chitosan for sports performance in life Quenching dry mouth e.
In many extraterrestrial environments, including Mars, we would bacteri amino acids syynthesis addition bactreia other organic compounds to be available to any Amibo biota. Pathsay are delivered to Aminl planetary surface in carbonaceous chondrite meteorites Cronin and Batceria ; Pathwa et syntheais.
For example, a wide range of amino acids Citrus fruit cultivation been detected in paghway chondrites.
The frequency ptahway the prebiotic Amini of amino acids and their abundances follow thermodynamic bactdria with the chemically simple compounds being most abundant Miller, ; Pizzarello, ; Pizzarello abcteria Shock, ; Glavin et al. A large number of microorganisms bwcteria use amino acids pahtway electron donors for bcateria respiration or in fermentation Nixon et al.
In this process, they degrade the amino acid Aino. Microorganisms are unlikely to degrade amino acids bacteeia the bactria rate compared to degradation by abiotic processes. Rather, they will degrade the molecules according to their metabolic pathways, the accessibility of certain amino acids, the availability of other metabolizable organic compounds, and other organism-specific effects.
Thus, we could hypothesize that, in the process of degrading abiotic amino acids, microorganisms would leave a biosignature by the preferential degradation of certain amino acids in the environment around them.
This biosignature might be superimposed on the biosignature of the amino acids in the organism itself and that were synthesized by the organism, but it would be a distinct and additional biosignature reflecting non-random biological destruction of the abiotic amino acid pool. One attraction of such a biosignature is that, if cells alter the amino acid concentration in the environment around them, then, particularly in low biomass environments, that signature might be much more pervasive and easier to detect than the amino acid signature of the cells themselves, which could be highly localized and poorly preserved.
Furthermore, although this signature would assume the presence of amino acid-using life, the decrease or increase in any amino acids away from the expected background abiotic concentration could be an agnostic signature of metabolic processes.
In this study, we tested this hypothesis by investigating the metabolic usage of seven amino acids previously detected in both terrestrial environments and Martian meteorites by two distinct anaerobic microbial communities from Martian analog environments.
We used two distinct communities to determine if there were common patterns of degradation of certain amino acids that could potentially suggest a universal signature of amino acid degradation by life.
The almost complete degradation of glycine was common to both communities. For other amino acids, we observed different patterns of degradation with increased extracellular concentrations of some amino acids. We discuss the implications of these findings for life detection.
The samples investigated were collected in the frame of the MASE Mars Analogs for Space Exploration project, a 4-year collaborative research project supported by the European Commission Seventh Framework Contract.
The aim of the project was to characterize Mars analogue environments on Earth with regard to habitability and the search for potential biosignatures of extraterrestrial environments Cockell et al.
Samples analyzed herein were collected from two sulfidic springs close to Regensburg, Germany, where many sulfide-containing springs emanate from Mesozoic karst formations.
The two springs, in the Sippenauer Moor SM and Islinger Mühlbach ISM areas The sites SM and ISM are independent and not connected in the deep subsurface. Detailed analyses of the site microbiomes are already available Moissl et al.
All samples were taken under anoxic conditions for a detailed description see Cockell et al. Cultivation was performed under anoxic conditions. Samples from SM and ISM were inoculated in anoxic MASE medium II and supplemented with a mixture of proteogenic and non-proteogenic amino acids.
MASE II medium contains per liter: NH 4 Cl 0. Prior to inoculation, the medium was supplemented with a filter-sterilized amino acid broth. Amino acids such as alanine, aspartic acid, glutamic acid, glycine, leucine, serine, and valine are common in both biological samples and for example, carbonaceous chondrite meteorite Cronin and Pizzarello, ; Shimoyama et al.
Based on these data, the following mixture of amino acids was added to the medium: glycine, L-alanine, β-alanine, L-aspartic acid, DL-proline, L-leucine, L-valine, L-phenylalanine and L-isoleucine Table 1.
The amino acid broth used in this study included some proteinogenic amino acids that are most likely not found in meteorites due to their complexity in synthesis.
The final concentration of each added amino acid was 10 mM, and the pH was adjusted to 7. One millilitre of the environmental sample was added to 20 ml of medium and incubated at 30°C.
A negative control NCi. TABLE 1. Chemical properties of the supplemented amino acids and their side chains. After defined time points, 1.
The first sample was taken immediately after inoculation T0followed by samples after 7, 14, 28, 56, and 90 days of incubation. The sample was sterilized using a 0. Amino acid extraction was performed using a simplified procedure described in Aerts et al.
Therefore, the extraction protocol is described in the following only briefly. For sterilization, all glassware, including the columns with glass wool for amino acid extraction, were double wrapped in aluminum foil and placed into a furnace at °C for a minimum of 3 h.
A sequential washing with basic-neutral-acid-basic solutions was made to activate the resin active sites. After the sequential washing procedure, 1. The sample was vortexTed at 2, rpm for 30 s and subsequently added to the column.
Note, that this first elution was not collected for further analysis and was disposed. The system used for amino acid analysis is described in Aerts et al.
Measurements were performed using an Agilent LC-MS system equipped with an ultraviolet UV and fluorescence FL detector system, an autosampler module where the amino acid derivatization is performed, and a MS ion trap mass spectrometer with electrospray ionisation. The column used for analysis was a × 3 mm 2.
The MS was operated in positive mode with optimised conditions for each individual amino acid. Amino acids were derivatised using a method based on Nimura and Kinoshita which was then automated in order to increase the robustness of the method. This automation was achieved by programming the autosampler module Agilent GB of the HPLC to mix the various reagents.
The approach used was as follows: the amino acid sample was mixed in a ratio with 0. In a typical measurement run, amino acid samples from one time point including negative control were analysed sequentially, including wash procedures and the analysis of amino acid standard solutions Agilent, part number: — Proline was not measurable as it cannot be derivatized and is therefore not detectable using the applied method.
Standards were run at the beginning and end of each run in order to track reagent degradation and system performance.
The standard deviation was added as error bars to the measurements of the amino acids of SM and ISM. FIGURE 1. Degradation of glycine from the two different enrichments A Islinger Mühlbach ISMand B Sippenauer Moor SM spiked with a broth of amino acids final concentration of each added amino acid was 10 mM over a time of 3 months.
C Negative control, i. FIGURE 2. LC-MS measurements of the three amino acids β-alanine, L-aspartic acid, and L-phenylalanine from the two different enrichments over a time of 3 months.
A Islinger Mühlbach ISMand B Sippenauer Moor SM spiked with a broth of amino acids final concentration of each added amino acids was 10 mM.
FIGURE 3. LC-MS measurements of the four amino acids L-alanine, L-valine, L-leucine, and L-isoleucine from the two different enrichments over a time of 3 months.
A Islinger Mühlbach ISMand B Sippenauer Moor SM spiked with a broth of amino acids final concentration of each added amino acid was 10 mM. Using LC-MS measurements, we investigated the differences in the amino acid distribution in the medium of two different microbial enrichments and the negative control.
We found that in the control samples in which no microbiota was added, no significant changes of amino acid concentrations were observed over time Figures 1 — 3. Therefore, the changes observed in the inoculated samples are attributed to microbial activity.
Note, because the signal of measured amino acids in NC for time point 56 days was significantly lower in comparison to the signal of the NC of the other time points, NC of time point 56 days was not considered for analysis. No such decrease was observed for the measurements of amino acids in the SM and ISM samples of the same time point, which points to a sample problem and not to an instrument malfunction.
The results after analysis using LC-MS revealed that the quantities of the non-glycine amino acids varied over time depending on the microbial community and the amino acid see Figures 1 — 3. Although the concentrations of amino acids varied between the two enrichments, we found one amino acid characteristic that was consistent with both enrichments Figure 1.
The depletion follows an exponential decay; see fit to the date in panel B of Figure 1.
: Amino acid synthesis pathway in bacteria| Our microbes not only produce antibiotics, they also overproduce amino acids | Link: Normal Module. Background color Organism. ID search. Module Complete only Including 1 block missing Including any incomplete. Reaction module Reaction modules Carboxylic acid metabolism 2-Oxocarboxylic acid chain extension RM 2-Oxocarboxylic acid chain extension by tricarboxylic acid pathway 2-Oxocarboxylic acid chain modification RM Carboxyl to amino conversion using protective N-acetyl group basic amino acid synthesis RM Carboxyl to amino conversion without using protective group RM Branched-chain addition branched-chain amino acid synthesis. We also observed a phenylalanine-specific T-box site upstream of the pheA gene in D. TRAP binding sites in the leader regions of the trp and pabA genes in the Bacillus spp. Start codons of the pabA genes are underlined. Newly identified TRAP sites are indicated by asterisks. subtilis , whose translation is regulated by the TRAP protein. We found orthologs of the yhaG gene in B. stearothermophilus , C. acetobutylicum and C. No homologs of yhaG were observed in the genomes of E. faecalis and S. pyogenes that lack the tryptophan biosynthesis pathway, and thus should transport tryptophan from the environment. We identified tryptophan-specific T-boxes upstream of the yhaG orthologs in both Clostridium species; in B. stearothermophilus the upstream region of this gene is unavailable. Thus, yhaG is regulated in B. subtilis and Clostridium at the RNA level by two different mechanisms, tryptophan-mediated TRAP repression and tryptophan-specific T-box antitermination, respectively. Analyzing the predicted T-box regulatory sites and positional gene clustering we identified a new candidate tryptophan ABC transporter, named trpXYZ , in the genomes of S. mutans , S. pyogenes , S. equi , E. faecalis , E. faecium , B. stearothermophilus , D. hafniense , B. cepacia , and M. loti the latter two are α-proteobacteria. The genes in the S. pneumoniae genome are SP , SP , SP hafniense has three trpXYZ paralogs, and two of them have tryptophan-specific T-boxes in the upstream regions. Additionally, trpXYZ is preceded by a tryptophan-specific T-box in S. Moreover, trpXYZ is located in one candidate operon with the ortholog of the kynU gene in M. kynU encodes l -kynurenine hydrolase, which catalyzes conversion of l -kynurenine into anthranylate Fig. Thus co-induction of the trpXYZ-kynU operon in tryptophan-depleted conditions leads to the transport of tryptophan from the medium and the concurrent accumulation of anthranylate, a tryptophan biosynthetic precursor. Additionally, trpXYZ is co-localized with the aroD gene in E. These pieces of evidence allow us to ascribe tryptophan specificity to all but one major clades of the trpXYZ family members on the phylogenetic tree Fig. Note that this assignment fills the above-mentioned gap in the E. pyogenes metabolic maps. Filled ovals: genes that are either regulated by tryptophan-specific T-boxes or positionally clustered with genes involved in tryptophan metabolism. Empty ovals: genes from the genomes that lack the tryptophan terminal pathway and thus should transport tryptophan from the environment. We predict tryptophan specificity for all but one major clades the latter is circled by a dotted line that contain genes with evidence for tryptophan specificity. We identified another candidate tryptophan transporter in B. Four members of the sodium transporter family homologous to yocR and yhdH in B. subtilis are present in this bacterium and two of them are regulated by the T-box antitermination mechanism. We assigned specificities based on the T-box regulatory elements. We predict that one of these genes, named here sdt1 , encodes a tryptophan-specific transporter, and the other gene, sdt2 , is serine-specific. Homologous genes of this transporter family were identified in the genomes of B. halodurans , B. aureus , L. monocytogenes and S. Additionally, a homologous transporter in Haemophilus ducrei forms an operon with genes of the tryptophan biosynthesis. The ycz-ycbK operon of B. subtilis is known to be regulated by TRAP-mediated repression and tryptophan-specific T-box antitermination [20]. A TRAP site and a tryptophan-specific T-box are located in the intergenic region of the yczA-ycbK operon and in the leader region of the yczA gene respectively. The yczA gene is known to encode the anti-TRAP protein, an inhibitor of TRAP activity [20] , but the function of YcbK is unknown. As this protein has a predicted 10 transmembrane segments and the gene ycbK is likely regulated by tryptophan, YcbK can be involved in transport of tryptophan or tryptophan-related compounds. The single-copy proteins in the Bacillus spp. are bi-functional. One could suggest that originally there existed a bi-functional protein, which had been independently duplicated in B. anthracis , L. aureus , and D. However, our analysis demonstrates that the only copy of the enzyme in the genomes of C. pyogenes , and S. pneumoniae is likely mono-functional either folate- or tryptophan-specific. Thus, the assumption of universally distributed bi-functionality would require too many independent duplication and loss-of-function events. The corresponding genes were co-localized with other genes of the respective pathways. This situation is still conserved in the genomes of B. However, some species have eliminated one of the pathways e. faecalis has eliminated both pathways, and accordingly, it has no representatives of this family. At the time of the B. halodurans branch formation, the trpG gene was lost from the trp operon, and the remaining PabA protein encoded in the pab operon acquired additional function in tryptophan biosynthesis, thus becoming bi-functional. Simultaneously, the pabA gene acquired the TRAP-dependent regulation of translation. We propose that TRAP bound to the upstream region of pabA blocks de novo initiation of pabA translation, whereas it does not prevent the ribosomal re-initiation at the pabA start codon, as the ribosome coming from the upstream pabB gene removes TRAP from the RNA. In this way, the tryptophan-dependent regulation of the bi-functional pabA gene does not interfere with folate synthesis. However, the cores of regulons in Gram-negative and Gram-positive species coincide. These include the trp operon, DAHP synthase and shikimate kinase genes, and the phhA gene. Interestingly, even the type of regulation of these genes is almost conserved: in both groups the trp operon is regulated at the RNA level although it is additionally regulated by a DNA binding repressor in γ-proteobacteria , whereas the DAHP synthase and shikimate kinase genes are regulated at the DNA level. In contrast, group-specific members of regulons, e. transporters yhaG , trpXYZ , mtr , tyrP , aroP , are regulated by variable mechanisms: by DNA-dependent regulation in Gram-negative genomes, and by RNA-dependent regulation in the Gram-positive group. The same pattern of regulation was observed for the phhA gene. One notable exception to this rule is provided by B. In contrast to other bacilli that display DNA level regulation of the aroA and aroF genes, B. anthracis has acquired T-boxes upstream of both genes, and thus shifted to RNA level regulation. The most general one is the T-box-dependent transcriptional regulation, which is present in all studied species. Another type of RNA-dependent transcriptional regulation, TRAP-mediated regulation, is unique to the Bacillus group except for B. anthracis , which lacks the TRAP protein. stearothermophilus , TRAP regulates transcription of the trp operon, which is regulated by tryptophan-specific T-boxes in all other species. stearothermophilus , where it appears to regulate transcription of DAHP synthase and chorismate synthase genes. anthracis , the same genes are regulated by tyrosine-specific T-boxes. Finally, in S. mutans , and L. lactis , DAHP synthase and shikimate kinase genes seem to be under transcriptional regulation by ARO boxes identified in this study. However, we could not identify the transcription factor responsible for this regulation, as no candidate sites or RNA elements were observed upstream of genes encoding proteins with potential DNA binding domains. This means that, unlike TrpR and TyrR of γ-proteobacteria, these hypothetical factors are not subject to auto-regulation. We are grateful to Dmitry Rodionov and Andrey Osterman for useful discussion. This study was partially supported by grants from the Howard Hughes Medical Institute and the Ludwig Institute for Cancer Research CRDF RBO Pittard A. In: Escherichia coli and Salmonella. Cellular and Molecular Biology Neidhardt F. et al. ASM Press , Washington, DC. Google Scholar. Google Preview. Sarsero J. Merino E. Yanofsky C. Otwinowski Z. Schevitz R. Zhang R. Lawson C. Joachimiak A. Marmorstein R. Nature , — Davidson B. Jackson E. Keller E. Calvo J. USA 76 , — Terai G. Takagi T. Nakai K. Genome Biol. Babitzke P. Gollnick P. Henkin T. Panina E. Vitreschak A. Mironov A. Gelfand M. Schneider T. Stormo G. Gold L. Ehrenfeucht A. Vitreshchak A. The RNApattern program: searching for RNA secondary structure by the pattern rule. Proceedings 3rd Int. Delorme C. Ehrlich S. Renault P. Grundy F. Haldeman M. Hornblow G. Ward J. Chalker A. Vinokurova N. Moscow 34 , — Thompson J. Gibson T. Plewniak F. Jeanmougin F. Higgons D. Nucleic Acids Res. Felsenstein J. Methods Enzymol. Green J. Nichols B. Matthews R. Valbuzzi A. subtilis regulatory protein TRAP by the TRAP-inhibitory protein, AT. Science , — Oxford University Press is a department of the University of Oxford. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide. Sign In or Create an Account. Advertisement intended for healthcare professionals. Navbar Search Filter FEMS Microbiology Letters This issue FEMS Journals Microbiology Books Journals Oxford Academic Mobile Enter search term Search. FEMS Journals. Advanced Search. Search Menu. Article Navigation. Close mobile search navigation Article Navigation. Volume Article Contents Abstract. Journal Article. Regulation of biosynthesis and transport of aromatic amino acids in low-GC Gram-positive bacteria. Panina , Ekaterina M. Graduate Program in Molecular, Cellular and Integrative Life Sciences, Molecular Science Building, University of California at Los Angeles, Los Angeles, CA , USA. Oxford Academic. Alexey G. Integrated Genomics, P. Box , Moscow , Russia. Institute for Problems of Information Transmission, Russian Academy of Science, Bolshoj Karetny per. Andrey A. Mikhail S. Revision received:. PDF Split View Views. Cite Cite Ekaterina M. Select Format Select format. ris Mendeley, Papers, Zotero. enw EndNote. bibtex BibTex. txt Medlars, RefWorks Download citation. Permissions Icon Permissions. Close Navbar Search Filter FEMS Microbiology Letters This issue FEMS Journals Microbiology Books Journals Oxford Academic Enter search term Search. Abstract Computational comparative techniques were applied to analysis of the aromatic amino acid regulon in Gram-positive bacteria. Aromatic amino acid , Regulation , T-box , TRAP , ABC transporter. Open in new tab Download slide. At the next step, a recognition rule was generated. If some regulatory sites had already been identified in experiment, a profile was constructed using the alignment of these known sites. If there were no known sites, an iterative procedure was performed in order to construct a profile. All L -mer words were selected in each upstream region. Each word was compared to all words in other regions, and one word, closest to the initial one, was selected in each region. These words were used to construct a profile. Thus we obtain as many profiles as there were words in the sample. Positional nucleotide weights in the profile were defined as:. The obtained profiles were used to scan the set of words again, and the procedure was iterated until convergence. Then the best profile was selected to be used as the recognition rule. The quality of a profile was defined as its information content [12] :. Listeria monocytogenes B aroF-aroB-aroH-hisC-tyrA-aroE ; T-trp T-trp trpE-pabA-trpD-trpC-trpF-trpB-trpA ; aroD-aroC ; pheA ; aroI ; aroA T —? Open in new tab. mutans aroI ATGGGGGCtaAgAT 26 S. Escherichia coli and Salmonella. Cellular and Molecular Biology. Google Scholar Crossref. Search ADS. A Bacillus subtilis gene of previously unknown function, yhaG , is translationally regulated by tryptophan-activated TRAP and appears to be involved in tryptophan transport. TyrR protein of Escherichia coli and its role as repressor and activator. The region between the operator and first structural gene of the tryptophan operon of Escherichia coli may have a regulatory function. Alternative secondary structures of leader RNAs and the regulation of the trp , phe , his , thr , and leu operons. Prediction of co-regulated genes in Bacillus subtilis on the basis of upstream elements conserved across three closely related species. Google Scholar OpenURL Placeholder Text. Posttranscriptional initiation control of tryptophan metabolism in Bacillus subtilis by the trp RNA-binding attenuation protein TRAP , anti-TRAP, and RNA structure. Google Scholar PubMed. OpenURL Placeholder Text. Computer prediction of RNA secondary structure. Regulation of expression of the Lactococcus lactis histidine operon. The Staphylococcus aureus ileS gene, encoding isoleucyl-tRNA synthetase, is a member of the T-box family. Inferring phylogenies from protein sequences by parsimony, distance, and likelihood methods. Google Scholar Google Preview OpenURL Placeholder Text. Inhibition of the B. Issue Section:. Download all slides. Views 3, More metrics information. Total Views 3, |
| ORIGINAL RESEARCH article | We found that E. faecalis lacks members of this family; the Bacillus , Streptococcus , and Clostridium genomes, excluding only B. lactis , L. monocytogenes , S. aureus , B. anthracis , and D. hafniense each have two paralogous genes. Moreover, out of two paralogs, one always lies within the trp operon, while the other co-localizes with the pab operon Fig. This suggests that the first class of paralogous genes trpG LL , trpG LM , trpG SA , trpG BQ, DH , see appendix is specific for tryptophan biosynthesis, whereas the second class of paralogs pabA LL , SAV , lmo , pabA DH is specific for folate biosynthesis. Squares: one paralog per genome; ovals: two paralogs per genome. Filled frames: genes located within the pab operons; empty frames: genes located within the trp operons. Italics: folate-specific enzymes; underlined: tryptophan-specific enzymes the specificity is identified in this study ; italics and underlined: bi-functional enzymes. We found that the single member of this family from C. acetobutylicum is localized in the trp operon in the genome and clustered with the tryptophan-specific paralog from B. anthracis in the tree. Besides, C. acetobutylicum lacks other pab genes. Thus, we propose tryptophan-related specificity rather than bi-functionality for this single protein. A similar situation was observed in C. difficile : it has a single gene, positioned in the pab operon, and clustered with folate-specific paralogs in the tree. Thus, we propose folate specificity for the protein from C. The unfinished genome of C. difficile lacks the trp operon. The complete genome of S. Thus we suggest that it is folate-specific. In contrast, the complete genome of S. pneumoniae and the partial genome of S. Thus we suggest that they are tryptophan-specific. Pairs of DAHP synthase genes of S. lactis , encoding proteins homologous to DAHP synthases AroA from Gram-negative bacteria, form gene clusters in S. mutans , but are located separately in L. mutans , and upstream of both DAHP synthase genes in L. Moreover, a similar sequence was found in the upstream regions of the shikimate kinase genes aroI in all three species Table 2. Notably, the reactions catalyzed by shikimate kinase and DAHP synthase are the only two irreversible steps within the common pathway of the biosynthesis of aromatic amino acids, and only these genes of the common pathway are regulated at the transcriptional level in γ-proteobacteria. Thus, we propose that the new conserved signal sequence plays a role in transcriptional regulation of the DAHP synthase and shikimate kinase genes in the genomes of Streptococcus and L. A new DNA signal regulating DAHP synthase and shikimate kinase genes of several species identified in this study. Position is given relative to the translation start site. Paralogous aroG genes are numbered for convenience. We also constructed a profile based on the PCEs described in [8]. Using this profile we found a new candidate site ACTTAAccaCGTT upstream of the aroF gene in B. A number of new candidate T-boxes were found upstream of genes involved in aromatic amino acid biosynthesis Fig. Expression of the aroF and aroA genes is predicted to be regulated at the DNA level in B. halodurans , and B. stearothermophilus see above. In contrast, tyrosine-specific T-boxes were found upstream of these genes in B. anthracis Table 1. The aroA-aroF-hisC-tyrA-aroE locus in B. anthracis appears to be strictly regulated by the T-box antitermination mechanism, as two possible tyrosine-specific T-boxes are located upstream of the aroA gene and one more tyrosine-specific T-box is located upstream of the aroF gene. The aroF gene in E. coli is known to be regulated by the tyrosine-specific repressor TyrR [4]. Finally, a tyrosine-specific T-box was observed in B. anthracis upstream of the phhA gene. PhhA catalyzes conversion of phenylalanine to tyrosine Fig. Multiple alignment of newly identified T-boxes from Gram-positive bacteria. The columns represent 1 genome abbreviations as in Table 1 ; 2 gene names; 3 T-box specificities. The complementary stems of the RNA secondary structure and positions of the hairpins and conserved boxes are shown in the upper lines. Base-paired positions are indicated by the gray background. Conserved positions and non-conserved nucleotides are shown in bold and light font, respectively. Specifier codons are double-underlined. The trp operons are known to be regulated at the RNA level by two different mechanisms, TRAP-mediated repression in B. subtilis [9] and T-box antitermination in L. lactis [14]. Additional candidate TRAP binding sites were found upstream of the trp operons and the trpG genes in B. stearothermophilus Fig. Tryptophan-specific T-boxes were found upstream of the trp operons in B. anthracis , S. mutans , L. lactis , C. acetobutylicum , S. aureus , and L. Thus, TRAP-mediated regulation was observed only in three Bacillus species, B. subtilis [9] , B. stearothermophilus this study , whereas in other Gram-positive bacteria, including B. anthracis , only the T-box antitermination mechanism was detected. Moreover, the mtrB gene, which encodes subunits of TRAP, is present only in these three Bacillus genomes. Interestingly, B. anthracis has at least twice as many T-boxes as other Gram-positive bacteria A. Vitreschak, unpublished. We also observed a phenylalanine-specific T-box site upstream of the pheA gene in D. TRAP binding sites in the leader regions of the trp and pabA genes in the Bacillus spp. Start codons of the pabA genes are underlined. Newly identified TRAP sites are indicated by asterisks. subtilis , whose translation is regulated by the TRAP protein. We found orthologs of the yhaG gene in B. stearothermophilus , C. acetobutylicum and C. No homologs of yhaG were observed in the genomes of E. faecalis and S. pyogenes that lack the tryptophan biosynthesis pathway, and thus should transport tryptophan from the environment. We identified tryptophan-specific T-boxes upstream of the yhaG orthologs in both Clostridium species; in B. stearothermophilus the upstream region of this gene is unavailable. Thus, yhaG is regulated in B. subtilis and Clostridium at the RNA level by two different mechanisms, tryptophan-mediated TRAP repression and tryptophan-specific T-box antitermination, respectively. Analyzing the predicted T-box regulatory sites and positional gene clustering we identified a new candidate tryptophan ABC transporter, named trpXYZ , in the genomes of S. mutans , S. pyogenes , S. equi , E. faecalis , E. faecium , B. stearothermophilus , D. hafniense , B. cepacia , and M. loti the latter two are α-proteobacteria. The genes in the S. pneumoniae genome are SP , SP , SP hafniense has three trpXYZ paralogs, and two of them have tryptophan-specific T-boxes in the upstream regions. Additionally, trpXYZ is preceded by a tryptophan-specific T-box in S. Moreover, trpXYZ is located in one candidate operon with the ortholog of the kynU gene in M. kynU encodes l -kynurenine hydrolase, which catalyzes conversion of l -kynurenine into anthranylate Fig. Thus co-induction of the trpXYZ-kynU operon in tryptophan-depleted conditions leads to the transport of tryptophan from the medium and the concurrent accumulation of anthranylate, a tryptophan biosynthetic precursor. Additionally, trpXYZ is co-localized with the aroD gene in E. These pieces of evidence allow us to ascribe tryptophan specificity to all but one major clades of the trpXYZ family members on the phylogenetic tree Fig. Note that this assignment fills the above-mentioned gap in the E. pyogenes metabolic maps. Filled ovals: genes that are either regulated by tryptophan-specific T-boxes or positionally clustered with genes involved in tryptophan metabolism. Empty ovals: genes from the genomes that lack the tryptophan terminal pathway and thus should transport tryptophan from the environment. We predict tryptophan specificity for all but one major clades the latter is circled by a dotted line that contain genes with evidence for tryptophan specificity. We identified another candidate tryptophan transporter in B. Four members of the sodium transporter family homologous to yocR and yhdH in B. subtilis are present in this bacterium and two of them are regulated by the T-box antitermination mechanism. We assigned specificities based on the T-box regulatory elements. We predict that one of these genes, named here sdt1 , encodes a tryptophan-specific transporter, and the other gene, sdt2 , is serine-specific. Homologous genes of this transporter family were identified in the genomes of B. halodurans , B. aureus , L. monocytogenes and S. Additionally, a homologous transporter in Haemophilus ducrei forms an operon with genes of the tryptophan biosynthesis. The ycz-ycbK operon of B. subtilis is known to be regulated by TRAP-mediated repression and tryptophan-specific T-box antitermination [20]. A TRAP site and a tryptophan-specific T-box are located in the intergenic region of the yczA-ycbK operon and in the leader region of the yczA gene respectively. The yczA gene is known to encode the anti-TRAP protein, an inhibitor of TRAP activity [20] , but the function of YcbK is unknown. As this protein has a predicted 10 transmembrane segments and the gene ycbK is likely regulated by tryptophan, YcbK can be involved in transport of tryptophan or tryptophan-related compounds. The single-copy proteins in the Bacillus spp. are bi-functional. One could suggest that originally there existed a bi-functional protein, which had been independently duplicated in B. anthracis , L. aureus , and D. However, our analysis demonstrates that the only copy of the enzyme in the genomes of C. pyogenes , and S. pneumoniae is likely mono-functional either folate- or tryptophan-specific. Thus, the assumption of universally distributed bi-functionality would require too many independent duplication and loss-of-function events. The corresponding genes were co-localized with other genes of the respective pathways. This situation is still conserved in the genomes of B. However, some species have eliminated one of the pathways e. faecalis has eliminated both pathways, and accordingly, it has no representatives of this family. At the time of the B. halodurans branch formation, the trpG gene was lost from the trp operon, and the remaining PabA protein encoded in the pab operon acquired additional function in tryptophan biosynthesis, thus becoming bi-functional. Simultaneously, the pabA gene acquired the TRAP-dependent regulation of translation. We propose that TRAP bound to the upstream region of pabA blocks de novo initiation of pabA translation, whereas it does not prevent the ribosomal re-initiation at the pabA start codon, as the ribosome coming from the upstream pabB gene removes TRAP from the RNA. In this way, the tryptophan-dependent regulation of the bi-functional pabA gene does not interfere with folate synthesis. However, the cores of regulons in Gram-negative and Gram-positive species coincide. These include the trp operon, DAHP synthase and shikimate kinase genes, and the phhA gene. Interestingly, even the type of regulation of these genes is almost conserved: in both groups the trp operon is regulated at the RNA level although it is additionally regulated by a DNA binding repressor in γ-proteobacteria , whereas the DAHP synthase and shikimate kinase genes are regulated at the DNA level. In contrast, group-specific members of regulons, e. transporters yhaG , trpXYZ , mtr , tyrP , aroP , are regulated by variable mechanisms: by DNA-dependent regulation in Gram-negative genomes, and by RNA-dependent regulation in the Gram-positive group. The same pattern of regulation was observed for the phhA gene. One notable exception to this rule is provided by B. In contrast to other bacilli that display DNA level regulation of the aroA and aroF genes, B. anthracis has acquired T-boxes upstream of both genes, and thus shifted to RNA level regulation. The most general one is the T-box-dependent transcriptional regulation, which is present in all studied species. Another type of RNA-dependent transcriptional regulation, TRAP-mediated regulation, is unique to the Bacillus group except for B. anthracis , which lacks the TRAP protein. stearothermophilus , TRAP regulates transcription of the trp operon, which is regulated by tryptophan-specific T-boxes in all other species. stearothermophilus , where it appears to regulate transcription of DAHP synthase and chorismate synthase genes. anthracis , the same genes are regulated by tyrosine-specific T-boxes. Finally, in S. mutans , and L. lactis , DAHP synthase and shikimate kinase genes seem to be under transcriptional regulation by ARO boxes identified in this study. However, we could not identify the transcription factor responsible for this regulation, as no candidate sites or RNA elements were observed upstream of genes encoding proteins with potential DNA binding domains. This means that, unlike TrpR and TyrR of γ-proteobacteria, these hypothetical factors are not subject to auto-regulation. We are grateful to Dmitry Rodionov and Andrey Osterman for useful discussion. This study was partially supported by grants from the Howard Hughes Medical Institute and the Ludwig Institute for Cancer Research CRDF RBO Pittard A. In: Escherichia coli and Salmonella. Cellular and Molecular Biology Neidhardt F. et al. ASM Press , Washington, DC. Google Scholar. Google Preview. Sarsero J. Merino E. Yanofsky C. Otwinowski Z. Schevitz R. Zhang R. Lawson C. Joachimiak A. Marmorstein R. Nature , — Davidson B. Jackson E. Keller E. Calvo J. USA 76 , — Terai G. Takagi T. Nakai K. Genome Biol. Babitzke P. Gollnick P. Henkin T. Panina E. Vitreschak A. Mironov A. Gelfand M. Schneider T. Stormo G. Gold L. Ehrenfeucht A. Vitreshchak A. The RNApattern program: searching for RNA secondary structure by the pattern rule. Proceedings 3rd Int. Delorme C. Ehrlich S. Renault P. Grundy F. Haldeman M. Hornblow G. Ward J. Chalker A. Vinokurova N. Moscow 34 , — Thompson J. Gibson T. Plewniak F. Jeanmougin F. Higgons D. Nucleic Acids Res. Felsenstein J. Methods Enzymol. Green J. Nichols B. flavum , 3-deoxy- D -arabino-heptulosonate 7-phosphate synthase DAHPS is feedback inhibited concertedly by phenylalanine plus tyrosine and weakly repressed by tyrosine. Other enzymes of the common pathway Figure 3 are not inhibited by phenylalanine, tyrosine and tryptophan, but the following are repressed: shikimate dehydrogenase, shikimate kinase SK , and 5-enolpyruvylshikimatephosphate synthase. Elimination of the uptake system for aromatic amino acids in C. glutamicum resulted in increased production of aromatic amino acids in deregulated strains. Biosynthetic pathways for L -tryptophan, L -phenylalanine and L -tyrosine. DAHPS, 3-deoxy- D -arabino-heptulosonate 7-phosphate synthase; DQS, dehydroquinate synthase; SD, shikimate dehydrogenase SD ; SK, shikimate kinase SK ; CS, chorismate synthase; CM, chorismate mutase; TAT, tyrosine amino transferase; PD, prephenate dehydratase; AS, anthranilate synthase. flavum producer to azaserine resistance. Azaserine is an analog of glutamine, the substrate of anthranilate synthase AS. Such a mutant showed a fold increase in the activities of DAHPS, dehydroquinate synthase, shikimate dehydrogenase, shikimate kinase, and chorismate synthase. Another mutant, selected for its ability to resist sulfaguanidine, showed additional increases in DAHPS, dehydroquinate synthase and tryptophan production. The reason that sulfaguanidine was chosen as the selective agent involves the next limiting step after derepression of DAHPS, that is, conversion of the intermediate chorismate to anthranilate by AS. Chorismate can also be undesirably converted to p -aminobenzoic acid and sulfonamides which are p -aminobenzoic acid analogs. A sulfaguanidine-resistant mutant was obtained with C. The sulfaguanidine-resistant mutant was still repressed by tyrosine but showed higher enzyme levels at any particular level of tyrosine. Elimination of tryptophan permeases improved L -tryptophan production by E. A genetically engineered strain of E. A conventionally mutated strain of E. Gene cloning of the tryptophan branch and mutation to resistance to feedback inhibition yielded a C. The genes cloned were those which encode AS, anthranilate phosphoribosyl transferase, a deregulated DAHPS, and other genes of tryptophan biosynthesis. However, sugar utilization decreased at the late stage of the fermentation and plasmid stabilization required antibiotic addition. Sugar utilization stopped due to killing by accumulated indole. By cloning in the 3-phospho-glycerate dehydrogenase gene to increase production of serine, which combines with indole to form more tryptophan , and by mutating the host cells to deficiency in this enzyme, both problems were solved. L -Phenylalanine is another commercially important amino acid. It is used as food or feed additive. A deregulated strain of E. coli containing a feedback inhibition-resistant version of the chorismate mutase-prephenate dehydratase gene, a feedback inhibition-resistant DAHPS, and the O R P R and O L O L operator-promoter system of lambda phage. Further process development of genetically engineered E. Independently, genetic engineering based on cloning aroF and feedback resistant pheA genes created an E. DAHPS in the wild type was inhibited cumulatively by phenylalanine and tyrosine, whereas prephenate dehydratase was inhibited by phenylalanine. Similarly, C. L -Tyrosine is another aromatic amino acid, mainly utilized as a precursor in the synthesis L -3,4-dihydroxyphenylalanine, the preferred drug for the treatment of Parkinson's disease. Around metric tons of L -3,4-dihydroxyphenylalanine are produced every year via both enzymatic and chemical methods. L -Tyrosine over-production has been achieved by cloning shikimate kinase into a tyrosine-producing C. lactofermentum strain. glutamicum strain into the deregulated tryptophan producer, C. glutamicum KY a chorismate mutase-deficient strain, phenylalanine, and tyrosine double auxotroph with a desensitized AS. The use of E. coli for tyrosine over-production was achieved by replacing the pheLA genes of a phenylalanine-producing strain with a multi-gene cassette kanamycin resistance gene. Beta-alanine, also known as 3-aminopropionic acid 3-AP , is the sole naturally occurring beta-amino acid. It has been proposed as an intermediate in formation of acrylamide and acetonitrile or as a direct precursor of poly-beta-alanine nylon Poly-beta-alanine is used for cosmetics and water purification. Biological production of 3-AP has been carried out by whole cell conversion of beta-aminopropionitrile by Alcaligenes sp. Song et al. coli to convert glucose to 3-AP. This was the first report of glucose conversion to 3-AP. The overexpressed genes were aspA and panD. A review of the use of metabolic engineering to develop producers of L -arginine and its derivatives is that of Shin and Lee. High production of L -arginine has been achieved with metabolically engineered C. glutamicum at Improvement of L -arginine production was achieved by Zhang et al. Previously, C. glutamicum had been shown to be an improved producer of L -arginine. In Corynebacterium crenatum , N -acetyl- L -glutamate kinase NAGK catalyzes the second step in L -arginine biosynthesis and is inhibited by L -arginine. Study of NAGK synthesis in three mutants yielded improved activity, that is, higher specific activity and thermostability. Site-directed mutagenesis of the NAGK gene in C. crenatum yielded a strain producing crenatum strain SYPA-EH3, containing these mutations, plus one additional mutation, produced Further studies on overexpression, NADPH optimization and increased glucose consumption in fed-batch fermentations yielded a final strain producing Systems metabolic engineering further increased production to L -Ornithine is used as a food supplement and nutrition product. It is useful for liver diseases and wound healing, and increases serum levels of growth hormone and insulin-like growth factor It protects the liver by detoxifying excess ammonia in humans. Its use in foods is to reduce bitterness in juices and other beverages. It is usually produced by extraction from hydrolyzed protein or by microbial fermentation. Use of metabolic engineering to increase availability of NADH resulted in production of L -ornithine by C. The related compound putrescine is produced at Development of an L -citrulline process using C. glutamicum has been described by Hao et al. Citrulline is an intermediate in biosynthesis of L -arginine from L -glutamate in a seven-step pathway. By inactivating gene argG , encoding argininosuccinate synthetase converting citrulline to argininosuccinate, which normally is converted to arginine and gene argR , the repressor gene, strain CIT2 was obtained which produced 5. Overexpression of the argJ gene, encoding the enzyme deacetylating acetylornithine to ornithine which then is converted to citrulline , yielded strain CIT3 producing 8. L -Aspartic acid , a four carbon amino acid, is used to make the synthetic sweetener aspartame and amino analogs of C4 building block chemicals, for example, 1,4-butanol, tetra-hydrofuran and gamma-butyrolactone. Free and immobilized thermostable aspartase of B. subtilis YM, expressed in E. Gamma-aminobutyric acid GABA is used in pharmaceuticals and functional foods. It acts in mammalians as an inhibitory neurotransmitter to modulate the overall excitability of the central nervous system. It improves brain function, has anti-anxiety effects, tranquilizer effects, boosts fertility, has diuretic effects, anti-diabetic effects and is used in the treatment of epilepsy. It is also used to produce nitrogen-containing industrial chemicals, for example, N -methyl pyrrolidinone and bioplastics. A recombinant strain of C. glutamicum produced GABA when two glutamate decarboxylase genes gadB1 and gadB2 from Lactobacillus brevis were co-expressed. Important in this process was a urea supplementation strategy. Urea is important in production of the precursor L -glutamate, serving as a nitrogen source, and for maintenance of neutrality during glutamate production. Another strain of C. glutamicum produced Gene odha encodes the E1 subunit of the 2-oxoglutarate dehydrogenase complex; gene pyc encodes pyruvate carboxylase. The result was generation of a recombinant strain which accumulated GABA. A coupled process to produce both GABA and lactic acid was developed by Zhao et al. Bioconversion of L -glutamate to GABA by resting cells of L. brevis TCCC has been further reviewed by Shi et al. Genetic engineering of C. These are platform chemicals, that is, building blocks for bio-based plastics such as nylon, polyamides and polyesters. The pathway from glucose to 5-AVA includes lysine, the well-known product of C. glutamicum metabolism. Metabolic engineering of E. coli W led to production of It does not participate in protein synthesis. It can be used to induce the immune response to increase plant resistance to disease, as well as improving the growth of young pigs. Thus, it has potential applications as a fertilizer additive for crops and feed additive for stock farming. The techniques used in the metabolic engineering work were: blockage of competing and degradative pathways, overcoming the bottleneck of carbon flux to homoserine, deregulating feedback inhibition, increasing export flux, and modifying the TdcC transporter. Synthesis of L -pipecolic acid from DL -lysine was studied by Tani et al. coli strain carrying a plasmid encoding AIP apoptosis-inducing protein , Δ 1 -piperideinecarboxylase reductase Pip2C reductase and lysine racemase from Pseudomonas putida , and glucose dehydrogenase from B. High optical purity over L -pipecolic acid is a non-proteinogenic amino acid involved in synthesis of FL, rapamycin and other products. It is the most abundant free thiol. It is a redox-active tripeptide thiol and has the following activities: anti-oxidization maintaining redox balance , detoxification, immune booster. GSH is used in the pharmaceutical, cosmetic and food industries. Fermentation appears to be a favorable production process due to the mild conditions used, high recovery and low cost. A review of GSH biosynthesis is that of Yang et al. Thus, gamma- L -glutamyl- L -cysteine is formed from L -glutamate and L -cysteine, the rate-limiting step of GSH biosynthesis, and GSH is formed by L-GSH synthetase which connects gamma- L -glutamate- L -cysteine with glycine. GSH inhibits GSH synthetase, resulting in low fermentation levels. A strain of E. coli was engineered to contain this GSH synthetase. The recombinant E. coli strain coupled with S. cerevisiae led to the production of Enzyme Gsh F, which contains both γ-glutamylcysteine synthetase γ-GCS and glutathione synthetase GSH II activities from Streptococcus thermophilus , has been used for GSH biosynthesis by a recombinant strain of E. The strain produced two enzymes, one from Actinobacillus pleuropneumoniae and the other from Actinobacillus succinogenes. The microbial production of amino acids, through fermentation, serves a market with strong prospects of growth and contributes significantly to our quality of life. Also, some amino acids are proving very valuable as biosynthetic precursors for the manufacture of therapeutics. The ability of a fermentation process to produce an amino acid depends on the overproduction capacity of the strain being used. In the early years of fermentation processes, strain development depended entirely on classical strain breeding involving intensive rounds of random mutagenesis, followed by equally strenuous programs of screening and selection. However, recent innovations in molecular biology, on the one hand, and the development of new tools in functional genomics, transcriptomics, metabolomics and proteomics on the other, have resulted in more rational approaches for strain improvement. Most of the amino acids are usually produced by fed-batch processes using high performance mutants, and separation by ion exchange chromatography for crystallization. As can be seen from a good part of this article, the actinobacterium C. glutamicum has been very useful for production of amino acids. Its genome sequence has been elucidated and published. High throughput technologies, such as genomics, transcriptomics, proteomics and metabolomics, have been applied to study C. Its benefits are GRAS Generally Recognized As Safe status, rapid growth to high cell densities, genetic stability, a limited restriction modification system, lack of autolysis, maintenance of metabolic activity under growth-arrested conditions, low protease activity favoring recombinant protein production, plasticity of metabolism, strong secondary metabolism properties, broad spectrum carbon utilization pentoses, hexoses, and alternative carbon sources , and stress tolerance to carbon sources. Other products of C. The organism has also been useful for producing proteins because of a low level of extracellular protease activity and the presence of two native protein secretion mechanisms. The roles of amino acids and, in turn, microbial fermentations, stand to grow in stature, especially as we enter a new era in which the use of renewable resources is recognized as an urgent need. Becker, J. 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Charles A Dana Research Institute for Scientists Emeriti R. You can also search for this author in PubMed Google Scholar. Correspondence to Arnold L Demain. Dedication: We are delighted to dedicate this manuscript to Professor Hamao Umezawa, a great scientist and a great man. It is indeed a pleasure to have our contribution included as part of the Special Issue devoted to him. Reprints and permissions. Our microbes not only produce antibiotics, they also overproduce amino acids. J Antibiot 71 , 26—36 Download citation. Received : 13 July Revised : 28 September Accepted : 04 October Published : 01 November |
| REVIEW article | tuberculosis in the homodimer conformation. C Anthranilate phosphoribosyltransferase from M. tuberculosis PDB code 2BPQ. The two domains of each monomer, small and large, are colored red and blue, respectively, and the active site cleft is indicated by the bound benzamidine molecule, shown as spheres with carbon atoms colored yellow. Di Bifunctional E. coli phosphoribosyl anthranilate isomerase enzyme colored in blue and the indoleglycerolphosphate synthase domain colored red PDB code 1PII. The phosphate, shown as pink spheres, identifies the position of the phosphoribosyl anthranilate isomerase enzyme active site. Dii Dimeric monofunctional phosphoribosyl anthranilate isomerase from T. thermophilus PDB code 1V5X. E The heterotetrameric tryptophan synthase from M. tuberculosis PDB code 5TCF. The α subunits are colored in red, while the β subunits are colored in blue and cyan to highlight the subunit interface. Tryptophan is the most chemically complex and is the least abundant of the AAA. The biosynthesis of tryptophan occurs only in plants and microbes, and therefore contains multiple attractive targets for the development of herbicides and antimicrobials. Chorismate is the branching point from where the biosynthesis of tryptophan diverges from that of phenylalanine and tyrosine. The pathway from chorismate to tryptophan is shown in Figure 4. Figure 4. Synthesis of tryptophan from chorismate. After the seven-step pathway via shikimate generates chorismate, the biosynthesis of tryptophan diverges from those of the other two AAA. Tryptophan synthase A or α-subunit cleaves indole glycerolphosphate into indole and glyceraldehydephosphate, while tryptophan synthase B or β-subunit is responsible for the PLP-dependent condensation of the latter two compounds into tryptophan. The enzyme catalyzing the committed step of tryptophan biosynthesis is anthranilate synthase AS EC 4. AS, also designated as TrpG or TrpE, belongs to the lyase family, in particular to the oxo-acid-lyases capable of cleaving carbon-carbon bonds. It is allosterically inhibited by the final product of the pathway, tryptophan, and is an important player in partitioning in chorismate toward tryptophan biosynthesis. The formation of anthranilate involves the transfer of ammonia from the donor glutamine to chorismate, generating glutamate and pyruvate apart from anthranilate. This reaction is classified as a 1,4-nucleophilic substitution by ammonia followed by the elimination of pyruvate. Anthranilate synthase is considered to share evolutionary origins with other chorismate-metabolizing enzymes such as salicylate synthase EC 4. It has been shown that S. Another study demonstrated that only a few mutations were sufficient to induce AS activity in an aminodeoxychorismate synthase, which in its native form does not eliminate pyruvate Culbertson et al. Anthranilate synthase contains two components, AS-I TrpE , and AS-II TrpG , both EC 4. AS-I synthesizes the intermediate 2-aminodeoxyisochorismate ADIC from chorismate and ammonia Morollo et al. The enzyme mechanism and active chemistry are described in detail in another review Romero et al. Briefly, the AS-I subunits bind to aminate chorismate, when high concentrations of ammonia are present. The AS-II subunits releases ammonia for the amination of chorismate via the formation of a γ-glutamyl- S- cysteinyl enzyme intermediate. Magnesium is suggested to make the 4-hydroxyl group of chorismate a better leaving group. Anthranilate synthase organization and size differs between bacteria and plants. Some bacterial AS contain the two subunits, α and β Figure 3B , in various oligomeric compositions such as αβ, α 2 β 2 , or α 3 β 3 , or with a fusion of the two subunits Romero et al. Other bacterial AS such as the fused Streptomyces venezuelae are not only monomeric, but also cannot use ammonia instead of glutamine to aminate chorismate Ashenafi et al. Among pathogenic bacteria from which crystallographic structures of AS are available, the oligomeric organization differs substantially between Serratia marcrescens and S. typhimurium , where the α 2 β 2 tetramer associates via the AS-I subunits Spraggon et al. tuberculosis enzyme, AS-I is a homodimer, even when AS-II is present Bashiri et al. In the last enzyme, the allosteric binding site for the inhibitor tryptophan is found near the interfacial region. thaliana Niyogi and Fink, ; Niyogi et al. It is noteworthy that in some eukaryotes such as fungi, the AS complex may contain enzyme subunits with other functions in the tryptophan biosynthesis, i. In plants, such as potato and tobacco, as well as in bacteria, such as Pseudomonas putida , the presence of tryptophan-sensitive and -insensitive AS isozymes have been suggested as indicative of the existence of complete but distinct pathways for both primary and secondary metabolism of tryptophan Hrazdina and Jensen, It was reported that the second set of AS gene products in P. putida and P. aeruginosa , which are tryptophan-insensitive, participate in the biosynthesis of the blue-green phenazine pigment, pyocyanin Essar et al. Also known as anthranilate phosphoribosyl synthase APR synthase or TrpD EC 2. AnPRT is a member of the phosphoribosyl transferase PRT involved in nucleotide biosynthesis and salvage apart from AAA biosynthesis Sinha and Smith, A number of microbial AnPRT have been studied in detail, with the M. tuberculosis enzyme showing some interesting structural features Castell et al. In this case, an unusual channel, which could deliver anthranilate to the active site, has been observed. Multiple anthranilate-binding sites have been reported within this channel and may account for the substrate inhibition caused by anthranilate. The biochemical role of this substrate channeling may be to deliver the PRPP to anthranilate leading to phosphate attachment , instead of water which would lead to hydrolysis. AnPRT inhibitors based on these multiple binding sites have been explored Evans et al. AnPRT enzymes are homodimeric, with each subunit constructed from two domains that interact via a hinge region that contains the active site Figure 3C. Also known as PRA isomerase or TrpF EC 5. The enzyme architecture varies widely, with some bacteria such as E. coli containing TrpF fused to the C-terminus of the next enzyme in the pathway Wilmanns et al. Due to the existence of an analogous Amadori rearrangement in the biosynthesis of histidine catalyzed by the ProFAR isomerase EC 5. Indeed, a dual-functional enzyme called PriA has been discovered in M. tuberculosis and S. coelicolor Due et al. The bifunctional enzyme is monomeric with the C-terminal end facilitating the phosphoribosyl anthranilate isomerase reaction Figure 3Di. In contrast, the mono-functional enzyme is monomeric in mesophiles, whereas in thermophiles where increased structural stability is required, the phosphoribosyl anthranilate isomerase is observed primarily as a dimer Figure 3Dii. Regardless whether this reaction is catalyzed by a mono- or bi-functional enzyme, the domain responsible for the isomerization adopts the same basic βα 8 fold. The active site is located at the C-terminal end of the barrel and in dimeric structures, the loops located at the N-terminal side of the barrel interlock the monomers together. An increased helical content and increased numbers of charged residues observed in the thermophiles has also been proposed to contribute to enzyme stability. TrpC or indoleglycerol-phosphate synthase EC 4. In some bacteria such as E. coli , the IGP synthase is fused with the previous enzyme in the pathway, leading to a bi-functional PRA synthase: IGP synthase Wilmanns et al. In this enzyme, a conserved glutamate and two conserved lysine residues have been identified as essential for catalysis. A notable feature of the IGP synthase reaction is that it is a series of biochemical steps—condensation, decarboxylation and dehydration in that sequence, whose kinetic mechanisms have recently been elucidated Schlee et al. The final step of tryptophan biosynthesis Figure 4 is catalyzed by tryptophan synthase or TrpAB EC 4. The enzyme consists of a α 2 β 2 tetramer Figure 3E , as demonstrated by the recent report of the M. tuberculosis crystal structure Wellington et al. The α subunit generates indole from IGP by means of a retro-aldol type of reaction wherein glycerolphosphate is eliminated, and channels the indole into the second active site, which is present in the β subunit. The second active site chemistry involves a typical PLP Schiff base mechanism. The activation of the substrate serine replaces an active site lysine attached to PLP. Indole acts as a nucleophile to displace a water molecule and the elimination-addition ends by indole condensing with a 3-carbon unit to form tryptophan. The shielding of reaction intermediates from the bulk solution by tunneling between the active sites and the complex allosteric coupling of the bound subunits have been the subject of many structural and mutagenesis studies. NMR studies, in particular, have shed light on the intricate dance of the tryptophan synthase components Axe and Boehr, and an extensive account of the workings of tryptophan synthase nanomachine is available elsewhere Dunn et al. Since animals lack the AAA biosynthetic pathways, pathogens that biosynthesize AAA are attractive targets for developing new anti-infective substances, especially since the rise of antibiotic resistance threatens the effectiveness of traditional antimicrobials. An increase in the number of available structures of enzymes in protein structure databases solved by X-ray crystallography particularly enzymes from pathogenic microbes , combined with recent improvements in computational methods applied to elucidate and understand enzyme function as well as the increasing pace of genome sequencing and annotation of microbes in the last decade, have opened up enormous opportunities to develop new antimicrobial targets as well as to discover new antimicrobial molecules. The shikimate pathway furnishes not only the AAA, but also molecules such as vitamins and cofactors, and therefore is attractive for the discovery and or development of new chemotherapeutic agents Lamichhane et al. Enzymes involved in the shikimate pathway have been detected in the protists, Toxoplasma gondii which causes toxoplasmosis and one of the malarial parasites, Plasmodium falciparum Roberts et al. However, only the last enzyme of the pathway, chorismate synthase has been identified conclusively on the basis of genome annotation, and the remaining enzymes seem to be missing. Six of the seven enzymes of the pathway were identified in M. tuberculosis Cole et al. For Helicobacter pylori , the causative agent of gastric ulcers and a type I carcinogen, four of the seven shikimate pathway genes, aroQ, aroE, aroK , and aroC , are essential. pylori infections, and therapeutics based on inhibiting these two enzymes were reviewed extensively elsewhere González-Bello, SK phosphorylates shikimate at the 3-hydroxy group, at the expense of ATP. coli contains two types of enzymes SK1 AroK and SK II AroL , but most bacteria have only one SK variant. SK inhibitors have been developed both by a substrate-mimetic strategy as well as by screening compound libraries. The P-loop is conserved in many ATP- and GTP-dependent proteins. Therefore, substrate mimics of shikimic acid were designed to bind the substrate-binding SB site of SK specifically Blanco et al. Many of the compounds developed in that study for M. tuberculosis SK Mt-SK were reversible competitive inhibitors and some, such as the 3-aminoshikimates closely resembled the structure of shikimate. The inhibition kinetics and molecular modeling of these compounds showed that fixing the C4 and C5 hydroxyl groups in the diaxial conformation in the substrate mimic might be a good way to inhibit SK, due to the dramatic reduction in the flexibility of two domains— 1 the SB domain residues 9—17 in Mt-SK and 2 the LID domain residues — in Mt-SK. Arg was identified as a key residue during ATP-binding, product release and also a Lewis acid during catalysis. Other SK-targeting compounds have been discovered, for example, an LC-MS based screening of around compounds at the National Institutes of Health NIH Tuberculosis Antimicrobial Acquisition and Coordination Facility identified three inhibitors with sub-micromolar IC 50 values for Mt-SK Simithy et al. pylori SK Han et al. High-throughput virtual screening efforts have also been reported Segura-Cabrera and Rodríguez-Pérez, ; Coracini and de Azevedo, DHQ catalyzes the dehydration of 3-dehydroquinate to 3-dehydroshikimate, a reversible reaction. Two forms of DHQ with no similarity at the level of primary sequences, and differing biochemical and biophysical properties are found Kleanthous et al. DHQ1 AroD found in plants, fungi and some bacteria such as E. coli and Salmonella typhi , catalyzes a syn dehydration based on a Schiff base mechanism. Apart from biosynthesis, DHQ1 may also be required for virulence in some bacteria Racz et al. It is essential in prominent pathogenic species such as M. tuberculosis and H. pylori Database for essential genes. In Mt-DHQ2 M. tuberculosis DHQ2 , a conserved aspartate Asp 88 residue from an adjacent enzyme subunit triggers the conversion of an essential tyrosine Tyr24 into tyrosinate, while the positive charge of Arg19 stabilizes the enolate intermediate generated from dehydroquinate. The reaction was suggested to go through an enolate instead of an enol intermediate, due to the significantly lower energy of the enol intermediate Blomberg et al. Since the formation of the enolate involves abstraction of the C2 axial hydrogen by Tyr24, replacement of this hydrogen by another group is expected to inhibit the enzyme. However, substitution of the C2 equatorial hydrogen by benzyl groups also generates effective inhibitors as the methylene group permits close stacking of the benzene ring close to the aromatic ring of the Tyr 24 residue González-Bello et al. The reaction mechanism also entails a ring-flattening between the C2 and C3 positions during the elimination. Therefore, compounds mimicking the enolate transition state could be good competitive inhibitors and the first inhibitor synthesized and tested using this approach was 2,3-dehydroquinic acid Frederickson et al. It was shown to be a reversible competitive inhibitor of Mt-DHQ2 and Sc-DHQ2 S. coelicolor- DHQ2. The solution of the X-ray diffraction structure of Sc-DHQ2 with an inhibitor containing a C2-C3 double bond bound at the active site PDB entry 1GU1, 1. An important feature of DHQ2 substrate recognition is the carboxylate binding pocket which has been a challenge for in vitro optimization of anti-tuberculosis drugs, but an ester prodrug approach was demonstrated to improve antibacterial activity by increasing the cell permeability of mycobacteria Tizón et al. Several drug-like aromatic Mt-DHQ2 inhibitors with anti-tubercular activity in the micromolar range have been described, including some nitrobenzyl-gallate analogs González-Bello et al. TB is one of the great public health challenges and the need for new drugs, especially those with novel targets and modes of action Ma et al. This has generated interest in the metabolism of the TB pathogen. Although the complete genome sequence of Mycobacterium tuberculosis has been published Cole et al. The genes already identified in this context are also not arranged in a single open reading frame ORF and were therefore identified only after genetic complementation and biochemical studies. The work of the TB Structural Genomics Consortium TBSGC enabled the determination of crystal structures of many proteins from M. tuberculosis including those involved in tryptophan metabolism such as, TrpB, TrpC, and TrpE Lee et al. TrpD was shown to be necessary for colonization of the lungs Lee et al. Remarkably, a tryptophan auxotrophic strain was not virulent even in mice with severe combined immunodeficiency Smith et al. A decrease in tryptophan concentration and an increase in the activity of indoleamine-2,3-dioxygenase IDO EC 1. Therefore, it could be inferred that tryptophan degradation was induced specifically in the lungs by the host as a tryptophan starvation response to counter the pathogen. While plant tryptophan synthase inhibitors with sub-micromolar potency Sachpatzidis et al. A sub-micromolar inhibitor for TrpC was reported Shen et al. Curiously, inhibitors designed with one enzyme in the biosynthetic pathway as targets, often have activities against other enzymes in the pathway Kozlowski et al. One of the two putative anthranilate synthase genes in TB was found to be a salicylate synthase involved in mycobactin synthesis Harrison et al. The same authors showed further that TrpE knockout mutants of the bacterium were killed both in vitro and inside macrophages. Novel IGP synthase inhibitors that are effective against MDR TB have been reported Shen et al. Anthranilates fluorinated at the 5- or 6- position of the benzene ring were demonstrated to be antibacterial, with modest in vivo activity in mouse models Zhang et al. Since the fluoro-anthranilates were not shown to be TrpD inhibitors for the E. coli enzyme, but alternate substrates leading to formation of fluorinated IGP in vitro Cookson et al. Either, the biosynthesis is inhibited downstream of TrpD, or the fluorinated tryptophan formed at the end of this pathway is toxic to the bacterium. Incorporation of fluorine at the 4-, 5-, or 6- positions of tryptophan was shown to be toxic to E. coli , possibly due to detrimental effects on protein structure Brown et al. Complete phenylalanine degradation has not been reported in plants Mazelis, and phenylalanine hydroxylase homologs are absent in plants as far as the published data indicate, with no candidates found in the genome of the model plant Arabidopsis thaliana. However, a unique phenylalanine hydroxylase dependent on folate was discovered in non-flowering plants, which is localized in the chloroplast Pribat et al. Complete degradation of tyrosine in A. thaliana was shown to proceed by the same pathway as in mammals Dixon and Edwards, The transamination of tyrosine by TAT occurs yielding 4-hydroxyphenylpyruvate, which is then transformed by 4-hydroxyphenylpyruvate dioxygenase to homogentisate. Homogentisate in plants acts as the precursor for tocopherols such as vitamin E and plastoquinones. Further catabolic steps convert homogentisate via ring cleavage ultimately into fumarylacetoacetate. Hydrolysis of fumarylacetoacetate yields fumarate and acetoacetate, thereby linking tyrosine and fumarate metabolism, potentially both inside and outside of mitochondria. Although tryptophan has been shown to be a precursor for the synthesis of many secondary metabolites such as auxins, phytoalexins, glucosinolates, and alkaloids Radwanski and Last, , to date, the elucidation of the tryptophan degradation pathway s has not been reported in plants. In plants, phenylalanine and tyrosine are catabolized to generate anabolic precursors for the phenylpropanoid pathway. Phenylalanine can be converted to cinnamate by the enzyme phenylalanine ammonia lyase PAL EC 4. The expression of PAL-encoding genes are highly regulated by different biotic and abiotic stresses, and conditions which increases the requirement of the cell wall component lignin Anterola and Lewis, Cinnamate can be further metabolized to p -coumaroyl CoA, a central metabolite in the phenylpropanoid pathway, which are involved in mediating responses pertaining to biotic and abiotic stresses Dixon, ; Casati and Walbot, The phenylpropanoid pathway has been reviewed extensively elsewhere Boudet, ; Vogt, These compounds impart mechanical strength to plant cells, and also participate in pest deterrence, drought resistance, UV protection, disease resistance, pollen viability and so on Nair et al. Compounds such as lignans, lignins, cutin, suberin, catechins, sporopolleins, flavonoids, isoflavonoids, proanthocyanidins, aurones, phenylpropenes, stilbenes, alkaloids, and acylated polyamines are derived from this pathway and involved in plant defense. In addition, some of these compounds are involved in the synthesis of colorful pigments that are present in flowers and fruits Fraser and Chapple, Newer genome-based approaches, such as the creation of an extensive database for P superfamily genes CYPedia based on the microarray analysis of A. thaliana and the analysis of over 4, re-annotated genes predicted to be active in plant metabolism for co-expression with P genes, have been described recently Ehlting et al. Phenylalanine-derived volatile compounds are involved in plant reproduction and defense Dudareva et al. Phenylpropanoids, benzenoids, phenylpropenes, and nitrogenous aromatics are the major classes of volatiles in this context. Phenylalanine is converted to phenylacetaldehyde by oxidative decarboxylation Kaminaga et al. Apart from phenylacetaldehyde, other phenylalanine-based volatiles include phenylethylacetate, 2-phenylethanol, methylbenzoate, and isoeugenol Watanabe et al. Phenylalanine is also the precursor for a class of sulfur-containing secondary metabolites called phenylalanine glucosinolates Reichelt et al. Tyrosine, instead of phenylalanine, is the direct precursor of coumarate in the phenylpropanoid pathway in some plants Neish, ; MacDonald and D'Cunha, , where the enzyme responsible for the transformation is tyrosine-ammonia-lyase TAL EC 4. Tyrosine decarboxylase TyrDC EC 4. TyrDC is distributed across the plant kingdom and is involved in the biosynthesis of defense compounds such as glycosides Ellis, and alkaloids Leete and Marion, The induction of TyrDC was shown to be induced upon wounding or fungal elicitor treatment Kawalleck et al. In addition, recent studies using A. thaliana have demonstrated that TyrDC is involved in abiotic stress response especially during drought and exposure to high salt concentrations Lehmann and Pollmann, TyrDC in A. thaliana also feeds into the production of alkaloids as well as cell-wall hydroxycinnamic acid amides Facchini et al. Tyrosine also serves as the starting compound for the biosynthesis of tocochromanols DellaPenna and Pogson, ; Mène-Saffranè and Dellapenna, as well as plastoquinones Norris et al. The committed step of tocochromanol biosynthesis involves TAT, which converts tyrosine into p -hydroxyphenylppyruvate Norris et al. Tyrosine is the precursor for meta -tyrosine, a non-proteogenic amino acid found in fescue grasses. It has been hypothesized that meta -tyrosine can be incorporated into proteins instead of phenylalanine by eukaryotic phenylalanine-tRNA synthases Duchêne et al. The incorporation of meta -tyrosine can lead to wide range of plant growth defects including growth retardation and inhibition of root development Bertin et al. Tryptophan is the precursor to the family of auxins hormones Gibson et al. While indoleacetic acid IAA is the most abundant auxin, other indole-containing auxins such as; 4-chloro-indoleacetic acid 4-Cl-IAA , indole butyric acid IBA and indole propionic acid IPA are also important and have integral roles in plants. Asymmetric auxin distribution in response to environmental cues govern the form, shape, strength and direction of growth of all organs and the interactions between various organs Benkov et al. At least four pathways have been proposed for the production of IAA from tryptophan. It should be noted that the complete pathways for the degradation of IAA are still not elucidated Strader and Bartel, The two-step auxin biosynthesis pathway via indolepyruvate IPy , which is highly conserved throughout the plant kingdom and has been characterized in several monocot and dicot plants, is well known. The first step of this pathway is the elimination of the amino group from the AA by the tryptophan aminotransferase TAA EC 2. The latter compound then undergoes oxidative decarboxylation catalyzed by the YUC family of flavin monooxygenases to produce IAA. The enzymes of the transaminase-dependent pathway for IAA biosynthesis were characterized in vitro in the recombinant enzyme Stepanova et al. Recombinant TAA1 catalyzes the PLP-dependent transfer of an amino group from tryptophan to 2-oxoglutarate, yielding IPy and glutamate. Disruption of TAA genes not only abolishes IPA production, but also affects the metabolism of other α-ketoacids and amino acids. Recombinant YUC6 from A. thaliana was purified and shown to be a FAD-containing enzyme, wherein NADH reduces the bound FAD to FADH 2 , which then reacts with molecular oxygen to form the C4α- hydro peroxyflavin intermediate that is the actual oxidizing species Dai et al. Other routes that generate IAA from tryptophan include 1 the indoleacetaldoxime IAOx pathway, which contains the two cytochrome P enzymes CYP79B2 and CYP79B3 EC 1. In addition, a putative tryptophan-independent pathway of IAA biosynthesis directly from indole has been proposed Normanly et al. Enzymatic decarboxylation of tryptophan by the PLP-dependent TDC produces the indole alkaloid tryptamine, which is found in small amounts in many plants. It is deemed to be a feedstock compound for pathways involved in synthesis of terpenoid indole alkaloids TIA and those that influence growth and the microbiome. Expression of TDC and TYDC in transgenic tobacco depleted the pools of tryptophan and tyrosine respectively, but in addition also perturbed pathways not directly involving AAA, such as methionine, valine, and leucine biosynthesis Guillet et al. Tryptophan is also converted to compounds associated with plant-insect and plant-pathogen interactions known as the indole glucosinolates Halkier, , which are natural products containing thioglucose and sulfonate bound to the oxime derived from of the amino acid bound to an oxime function Halkier and Gershenzon, IAOx also feeds into the indole glucosinolate pathway via an oxime-metabolizing enzyme CYP83B1 Naur et al. Another major category of tryptophan-derived secondary metabolites are the phytoalexins Pedras et al. The major indolic phytoalexin is camalexin, which accumulates upon infection with pathogens or the action of abiotic elicitors Zhao and Last, ; Böttcher et al. In plants, chorismate is not only a precursor of the three AAA, but also the initial compound for the biosynthesis of folates, such as tetrahydrofolate or vitamin B9 Basset et al. et al. Therefore, the shikimate pathway could potentially be engineered to augment the synthesis of folates or vitamin K in crop plants. Another enzyme in the shikimate pathway, 5-enolpyruvylshikimatephosphate synthase EPSPS , is the target of the well-known herbicide, N -phosphonomethylglycine or glyphosate commonly referred to as Roundup ® , which is a mimic of PEP and competitively inhibits EPSPS, thereby reducing the carbon flux through the pathway Healy-Fried et al. Non-plant EPSPS are used to provide herbicide resistance in transgenic crops Duke and Powles, Being the basis for Roundup-Ready transgenic crops, EPSPS has received much research attention Singer and McDaniel, ; Smart et al. The biosynthesis of AAA and secondary metabolites derived from them are often elevated in infection responses Ferrari et al. Manipulation of these responses could help improve plant protection against bacterial disease. Invading bacteria trigger the transcription of pathways including AAA metabolism and pigment biosynthesis within 12 h of infection Truman et al. Salicylic acid SA is a plant defense compound that accumulates in leaves in response to local and systemic acquired resistance against phytopathogens Malamy et al. SA applied externally on plant surfaces alone is able to trigger enhanced resistance to pathogens in A. Although, SA was initially shown to be synthesized from phenylalanine via the PAL pathway, inhibition of this pathway did not prevent the synthesis of SA Mauch-Mani and Slusarenko, ; Coquoz et al. It was shown in further studies that chorismate was converted into isochorismate by isochorismate synthase ICS EC 5. Tryptamine production in transgenic tobacco was shown to severely inhibit the reproduction of whiteflies Thomas et al. Since this work was done with transgenic plants, the possibility exists for a generic TDC-based plant protection strategy against whiteflies. The role of meta -tyrosine in inhibiting the growth of competing plants by fescue grasses Bertin et al. Tryptophan is the precursor of serotonin, which has multiple functions in plants. Tryptophan is decarboxylated to tryptamine by TDC, which then undergoes hydroxylation by a cytochrome P monooxygenase, forming serotonin Schröder et al. In dry seeds, serotonin is a sink for ammonia which can be toxic. Serotonin is present in plant spines, such as those of stinging nettles and the pain caused as a result of contact with them Chen and Larivier, , may deter browsing animals from consuming the plants. Since serotonin also affects the gut of animals, plants produce it in seeds and fruits as a way to promote the passage of seeds through the animal digestive tract in order to aid seed dispersal Feldman and Lee, Serotonin is further metabolized into the growth regulator melatonin, which is also synthesized in response to various biotic and abiotic stresses, such as pathogenic fungi, toxins, soil salinity, drought and extreme temperature Arnao and Hernández-Ruiz, Tryptophan is a precursor of thioquinolobactin, an antifungal agent that protects plants against the pathogen Pythium debaryanum Matthijs et al. AAA obtained by animals from the diet can be broken down or converted into other necessary compounds, such as neurotransmitters see Figure 1. Phenylalanine is often converted into tyrosine in animals and both these AAA feed into the biosynthesis of neurotransmitters, such as L-3,4-dihydroxyphenylalanine L-DOPA , dopamine, epinephrine, and norepinephrine Figure 1. It is not surprising, therefore, that metabolic defects in animals genes related to AAA catabolism have significant effects on their health. We will limit this review to a description of key AAA catabolic pathways in animals, along with a brief general discussion of pathologies related to each AAA catabolic pathway. Complete AAA degradation pathways described for plants also occur in animals, whereby they break down phenylalanine and tyrosine from proteins for recycling. Alkaptonuria is an inherited disorder affecting this function, caused by non-functional and or suboptimal activity of the enzyme homogentisate 1,2-dioxygenase dioxygenase HGD EC 1. Tyrosinemia is an inherited disorder in a single pathway involving mutations in one of three distinct enzymes involved in tyrosine degradation—fumaroylacetoacetate hydrolase EC 3. This is a major pathway for the catabolism of phenylalanine and tyrosine catabolism in animals. Here, many enzymes have additional roles in the synthesis of multiple neuroactive substances. The trace amines include all the neurotransmitters and neuroactive intermediates in this pathway except for L-DOPA, dopamine, epinephrine adrenaline and norepinephrin noradrenaline. It enables the biosynthesis of the neurotransmitters phenylethylamine and N -methylphenylethylamine directly from phenylalanine, in addition to dopamine, octapamine, tyramine, N -methyltyramine, syneprhine, 3-methoxytyramine, epinephrine and norepinephrine either directly from tyrosine or from phenylalanine, which is hydroxylated to tyrosine. The physiological effects of these monoamine neurotransmitters are reviewed elsewhere Broadley, Phenylalanine is converted into tyrosine by phenylalanine 4-hydroxylase PheOH or PAH EC 1. Tyrosine hydroxylase converts tyrosine to L-DOPA, which is rate limiting for the synthesis of the catecholamines dopamine, epinephrine and norepinephrine. Tryptophan hydroxylase converts tryptophan into 5-hydroxy-L-tryptophan en route to serotonin. All the AAAH enzymes contain iron and catalyze AAA hydroxylation using tetrahydrobiopterin. They act as rate-limiting enzymes in their respective pathways Grenett et al. Detailed reviews of AAAH structural biology Flatmark and Stevens, , regulation Fitzpatrick, and AAAH-based therapeutic targets Waløen et al. PheOH deficiency causes phenylketonuria PKU in humans, which is an inborn error of metabolism attributed to a single gene defect Erlandsen et al. PKU leads to a deficiency of tyrosine, which is continuously produced from phenylalanine in many animals for the synthesis of the catecholamine and trace amine neurotransmitters. Untreated PKU can lead to seizures, intellectual disability, behavioral problems, and mental disorders Al Hafid and Christodoulou, Most of the more than mutations in this enzyme PAH DB 2 are linked to PKU, while a few different mutations have been identified among patients suffering from non-PKU hyperphenylalaninemia HPA. In humans, knockout mutations in PheOH are not lethal, but the loss of TyrOH is, with the victims dying at a late embronic stage or briefly after birth Flatmark et al. Only two TyrOH mutations were so far associated with disorders of the basal ganglia Knappskog et al. In later studies, the human TyrOH locus has also been linked to bipolar disorder Smyth et al. Even though their biochemical reaction mechanisms are the same and their substrate specificities are similar, they have different expression and regulation patterns, as well as different physiological roles McKinney et al. TPH1 synthesizes most of the serotonin in circulation and is expressed chiefly in the gastrointestinal tract, adrenal glands, kidneys, and the pineal gland. TPH2 however, occurs in the serotonergic neurons with wide distribution in various cortices in the brain Amireault et al. Each AAAH contains a non-heme iron center and a 6 R -L-erythro-5,6,7,8-tetrahydrobiopterin BH4 cofactor, and requires a dioxygen molecule during catalysis. The cofactor is oxidized to quinonoid dihydrobiopterin qBH2 , which is regenerated to BH4 by the NAD P H-dependent dihydropteridin reductase. The structure of human PheOH hPheOH has been solved and shows an active site which is very open to the solvent and to the binding of exogenous ligands Kappock and Caradonna, ; Fusetti et al. The negative potential and hydrophobic nature of the active site is considered to promote the binding of positively charged amphipathic molecules such as the actual substrates, pterin cofactors, and inhibitors such as catecholamines Hufton et al. The catalytic iron is situated at the entrance of the pocket containing the active site, with space enough for both the pterin cofactor and the substrate Hufton et al. A highly conserved motif 27 amino acids long has been proposed to govern the binding of the cofactor tetrahydrobiopterin Jennings et al. The competitive inhibition of PheOH and TyrOH by catecholamines has been investigated using binary complexes of the dimeric proteins with various catecholamines. The molecular basis for the inhibition has been proposed to be the binding of the inhibitors directly to the catalytic iron center via the bidentate coordination of the two hydroxyl groups Erlandsen et al. The regulatory domains have also been the subject of structural studies; the solution structure of the regulatory domain of TyrOH shows a core ACT domain similar to that found in PheOH. When isolated, this domain of TyrOH forms a stable dimer, whereas the corresponding domain in PheOH exhibits an equilibrium between the monomer and dimer, with dimer stabilization afforded by the substrate phenylalanine. This correlates well with the fact that TyrOH is regulated by the binding of catecholamines, while PheOH is regulated by the substrate binding to an allosteric site Fitzpatrick, Phenylalanine is converted to the neurotransmitter phenylethylamine by the PLP-dependent enzyme aromatic L-amino acid decarboxylase AADC or AAAD EC 4. Phenylethylamine undergoes N -methylation catalyzed by phenylethanolamine N -methyltransferase PMNT EC 2. After the conversion of phenylalanine to tyrosine by AAAH, the latter is decarboxylated by AADC to form tyramine. If instead of decarboxylation, tyrosine is rerouted via a second AAAH reaction, the product is L-DOPA. When L-DOPA is decarboxylated by AADC, dopamine is formed. A minor pathway leads from tyramine to dopamine, with the enzyme catalyzing the hydroxylation being brain CYP2D in humans Wang et al. Dopamine is methylated to 3-methoxytyramine by the action of catechol- O -methyltransferase COMT EC 2. Dopamine is hydroxylated at the aminoethyl side chain in an R -specific manner by DBH to yield the major neurotransmitter norepinephrine. After tryptophan is hydroxylated by AAAH to 5-hydroxy-tryptophan 5-HTP , following which AADC catalyzes the decarboxylation of 5-HTP into serotonin. In most animals, serotonin is found in the gastrointestinal tract gut , blood platelets as well as in the central nervous system. It has a variety of functions in the gastrointestinal and nervous systems, a detailed description of which can be found elsewhere Berger et al. Altered serotonin levels are involved in many diseases and disorders. In the liver, serotonin is oxidized by monomine oxidase to the corresponding aldehyde, which is further oxidized by aldehyde dehydrogenase to 5-hydroxyindoelacetic acid 5-HIAA , which is eliminated via urine. The amounts of serotonin and 5-HIAA are elevated in certain tumors and cancers. Serotonin is present in insect venoms, where it is the component responsible for causing pain to animals upon injection of these venoms Chen and Larivier, Pathogenic amoebae produce serotonin, which causes diarrhea in humans McGowan et al. Serotonin is converted by serotonin N- acetyl transferase to N -acetyl serotonin; methylation of N -acetyl serotonin by S -adenosyl methionine SAM -dependent hydroxyindole O -methyl transferase yields melatonin. Melatonin is a neuro-hormone with many functions such as antioxidant, sleep-wake regulator and immune system regulator. Tryptophan is also the source of the trace neurotransmitter tryptamine via an AADC catalyzed decarboxylation. All three decarboxylation reactions, namely, the conversion of phenylalanine into phenylethylamine, tyrosine into tyramine and tryptophan to 5-HTP have been considered to be catalyzed by the same enzyme, at least in animals. Species-specific differences between AADC produced by various organisms exist and studies in Drosophila demonstrated that different tissues may contain distinct AADC isoforms. It was shown that alternative splicing patterns from transcripts of the same gene caused the expression of tissue-specific variants Morgan et al. Deficiency of pyridoxine decreases AADC stability. Since, PLP is required for AADC catalysis, this is not surprising. However, the apoenzyme was found to degrade to a 20 times faster than the holoenzyme due to the involvement of a flexible loop covering the active site Matsuda et al. The loop is fixed to the active site in the holoenzyme with a PLP Schiff base ligand interaction and stabilized; it fits into the entrance of the active site, held by hydrophobic interactions with the substrate catechol ring. The flexible loop is expected to be stabilized in vivo by adopting a closed structure binding the substrate aldimine, whereas the apoenzyme does not bind the substrate, leading to its preferential proteolysis Matsuda et al. The catalytic mechanism of AADC has been postulated to involve two intermediates, a Michaelis complex followed by an external aldimine. A flexible region around the residue Arg is exposed before ligand binding and forms a Michaelis complex. This in turn causes a conformational change, and during the following transaldimination, a more dramatic conformational change occurs, forming an external aldimine Ishii et al. AADC deficiency is an inherited neuromuscular disorder in humans caused by a deficit of this enzyme. Patients show reduced catecholamine levels and elevated 3-O-methyldopa levels and have symptoms such as hypotonia, hypokinesia, and signs of autonomic dysfunction from an early age Pons et al. All these mutations reduce the turnover number of the enzyme and most also alter the tertiary structure, with several experimental approaches pointing to incorrect conversion of the apoenzyme to the holoenzyme as the cause of the pathogenicity in a majority of the cases Montioli et al. The most striking results are observed upon mutation of the residues His70, His72, Tyr79, Phe80, Pro81, Arg, and Arg, which map to a key loop structure participating in the switch of the apoenzyme to the holoenzyme Montioli et al. Melanins are pigments that in animals are responsible for the coloration of eyes, hair, skin, fur, feathers and scales. While higher animals use melanins mainly for protection from radiation and in the immune response, insects utilize them many purposes such as hardening the cuticle, pigmentation of the exoskeleton, wound healing and innate immune responses. Melanins are derived from L-DOPA, which as mentioned before is derived from tyrosine. Tyrosinase is a rate-limiting oxidase containing copper, which catalyzes two separate steps in melanin biosynthesis, the hydroxylation of a monophenol is the first reaction, followed by conversion of the o-diphenol to the corresponding o-quinone. The o-quinone, for example dopaquinone, is further metabolized to eumelanins and pheomelanins Solano, Dopaquinone combines with cysteine forming either 2- S -cysteinyl-DOPA or 5-cysteinyl-DOPA, both of which form pheomelanins via benzothiazine intermediates. In the eumelanin pathway, dopaquinone is converted to leucodopachrome, which is the parent compound for dopachrome. The next intermediate is either 5,6-dihydroxyindole DHI or 5,6-dihydroxyindolecarboxylic acid DHICA , both derived from dopachrome. Both are converted into quinone which eventually form the eumelanins. A comprehensive review of the biosynthesis of the melanin pigments in insects and higher animals has been published elsewhere Sugumaran and Barek, There are multiple types of albinism caused by melanin deficiency, linked to different genes, among which type 3 oculocutaneous albinism results from a single-gene inborn metabolic defect, in this case mutations in the tyrosinase enzyme. Albinism entails a partial or complete lack of pigmentation in the skin, hair, and eyes. Albinism in humans is commonly connected with a number of vision defects and the lack of skin pigmentation may lead to heightened susceptibility to sunburn and skin cancers. Melanin granules are essential in immune cells and therefore, albinism leads to lowered immune defense Kaplan et al. Albinism is also associated in some cases with deaf-mutism Tietz, All known mammalian dopachrome converting enzymes transform dopachrome into DHICA, whereas insect and invertebrate enzymes convert dopachrome into DHI. A D-dopachrome converting enzyme was first discovered in mammals Orlow et al. It was separated from the complex, characterized Aroca et al. DCT is known to contain zinc at its active site Solano et al. Whereas the binuclear copper is critical for oxygen activation in tyrosinase, dopachrome tautomerism does not need such chemistry. Therefore, it has been suggested that one zinc atom binds the quinonoid side of dopachrome and the other one to the imine and carboxyl groups Palumbo et al. This geometry is considered favorable to stabilize the quinone methide intermediate and allow a rearrangement to DHICA. The guanidium group of arginine has the ideal geometry for binding the carboxyl group of the substrate, which will stabilize the quinone methide intermediate and favor the isomerization reaction leading to DHICA production in DCT. Arg is modified to Gln in the slaty mutant and this single amino acid change is solely responsible for the drastic reduction in the enzyme activity of the enzyme Jackson et al. A dopachrome conversion factor distinct from DCT has been characterized from insects Aso et al. Mutation in this gene causes the color of the cuticle to be yellow to brown and hence the name Drapeau, DCDT is linked to the innate immune response of insects De Gregorio et al. DCDT activity is enhanced in response to microbial infections in Drosophila melanogaster De Gregorio et al. Unlike the DCT of mammals, DCDT exhibits promiscuous substrate specificity and attacks a number of L-dopachrome derivatives, while not converting D-dopachromes Sugumaran and Semensi, The insect enzyme decarboxylates some dopachrome derivatives but tautomerizes others. It is unclear if DCDT contains metal cofactors, and whether it lacks the critical arginine residue found in DCT. In theory, a carboxyl residue instead of the guanidium group from arginine could induce the elimination of a quinone methide from the active site after dopachrome isomerization and subsequent non-enzymatic decarboxylation. If DCDT does not need metals for catalysis, then the mechanism of DCT would also have to be revised. For these reasons, solving the three-dimensional of structure of DCDT is important and would be expected to lead to major advances in understanding the melanogenic enyzmes. This oxidative pathway for tryptophan degradation exists in both prokaryotes and eukaryotes and was first described by Kotake It is depicted on the right side of Figure 5. Defects in this pathway in humans are implicated in serious pathological manifestations, such as Huntington's disease Pearson and Reynolds, , Alzheimer's disease Ting et al. Figure 5. Pathways for tryptophan catabolism. The kynurenine pathways are found in different bacteria. Mammals degrade tryptophan mainly in the liver via 3-hydroxyanthranilate, while the branch proceeding via kynurenic acid is found in the brain. The other two pathways occur in gut bacteria; the generation of indole by the action of tryptophanase occurs in enteric bacteria, whereas indoleacetate and indolepropionate are produced via indolepyruvate in strict anaerobes, mostly Clostridia. Other pathways exist in Lactobacilli which convert tryptophan to indolealdehyde I3A. Tryptophan can also be transaminated to indolepyruvate via amino group transfer with 2-oxoglutarate or pyruvate. The pathway in both eukaryotes and prokaryotes starts with the oxidation of tryptophan into N -formylkynurenine in a heme-protein dioxygenase reaction. There are two separate heme-containing dioxygenases, typtophan-2,3-dioxygenase TDO EC 1. In mammals, the former enzyme is expressed mainly in the liver, while the IDO is found in the lungs, intestines, and brain. The next enzyme, kynurenine formamidase or arylformamidase EC 3. In eukaryotes, a mitochondrial NADPH-dependent flavoenzyme called kynureninemonoxygenase EC 1. In mammals, an alternative sink for kynurenine is the formation of KA by a transamination followed by a dehydration, catalyzed by the PLP-containing kynurenine aminotransferase KAT EC 2. There are four different isoforms termed KAT I, II, III and IV in mammals including humans, which all have broad, but distinct substrate specificities, and have been reviewed extensively elsewhere Han et al. KA is a non-competitive antagonist of glutamate receptors and therefore influences glutamate-mediated neurotransmission Kessler et al. Due to this, both KA accumulation and KA deficiency have been implicated in a variety of neuropathological conditions Schwarcz et al. Apart from these, KA is an endogenous ligand for the G-protein coupled receptor GPR35 that is mainly expressed in immune cells Wang et al. KA is also implicated in the control of cardiovascular function by affecting the appropriate areas in the medulla oblongata Colombari et al. Following the formation of 3-hydroxykynurenine, kynureninase or kynurenine hydrolase EC 3. Eukaryotes and many prokaryotes convert tryptophan into 3-hydroxyanthranilate by this reaction. After this stage, 3-hydroxyanthranilate is further cleaved by a non-heme iron enzyme, 3-hydroxyanthranilate 3,4-dioxygenase EC 1. This compound represents a branching point: it can be either catabolized via 2-aminomuconate Martynowski et al. This section covers AAA catabolism by microflora resident in animal guts, pathogens which infect animals, as well microbes that utilize AAA for the biosynthesis of antibiotics. While some prokaryotes share the 3-hydroxykynurenine version of the kynurenine pathway with eukaryotes including animals as discussed in the previous section, in bacteria belonging to the genus Pseudomonas , kynureninase preferentially acts directly on kynurenine, hydrolyzing it to anthranilate and alanine Hayaishi and Stanier, The pseudomonads subsequently convert anthranilate to catechol by the action of an NADPH-dependent non-heme iron enzyme called anthranilate-1,2-dioxygenase EC 1. Catechol is further degraded via β-ketoadipate in several steps into water and carbon dioxide. Nevertheless, the broad distribution and inducible nature of the enzymes of this pathway among bacteria and fungi suggests roles in catabolism and secondary metabolism. Several antibiotics can be derived from the kynurenine pathway, including sibirimycin, which arises from a modified pathway involving a methylation step Giessen et al. Tryptophan dioxygenase is also implicated in the production of quinomycin antibiotics via a β-hydroxy-kynurenine intermediate Hirose et al. The kynurenine pathway is involved in the interplay between host and pathogens during infections. The host imposes tryptophan limitation on invading microorganisms by degrading it via the kynurenine pathway, whereas the pathogens use the pathway to synthesize compounds necessary for their growth and metabolism. Anthranilate is a key molecule which participates in many of these interactions. aeruginosa synthesizes quinolone quorum sensing molecules from anthranilate, which are important for virulence. While anthranilate can be derived from many pathways including tryptophan biosynthesis, when P. aeruginosa is grown in rich media, the kynurenine pathway becomes the source for anthranilate Farrow and Pesci, Some pathogens, such as Chlamydia psittaci , have evolved to evade host-imposed tryptophan depletion by replacing the anthranilate synthase in the tryptophan biosynthetic operon by a kynureninase Wood et al. Infection with Toxoplasma gondii has been speculated as a factor in increasing incidence of schizophrenia; in mouse models, stimulation of the kynurenine pathway was observed and the levels of several pathway metabolites including 3-hydroxykynurenine, quinolinic acid, and kynurenic acid are elevated Notarangelo et al. During Helicobacter pylori infection in the human gastric mucosa, a specific up-regulation of IDO expression is observed, which regulates multiple helper T-cell lines, resulting in lowered gastric inflammation Larussa et al. The obligate intracellular pathogen, Anaplasma phagocytophilum , which causes one of the most common tick-borne diseases, has been shown to up-regulate the expression of a specific organic anion uptake protein and a KAT enzyme enhancing its survival in the arthropod vector Taank et al. Recent studies have demonstrated that the pathway branching from kynurenine to kynurenic acid involving KAT is induced in bacterial meningitis and the metabolites thus produced contribute directly to the pathology of the disease Coutinho et al. Aerobic, microaerophilic and strictly anaerobic microorganisms occupy the gut or gastro-intestinal GI tracts of animals including humans. Prominent GI tract bacteria which utilize AAA as substrates include lactobacilli, enteric bacteria and strict anaerobes mostly Firmicutes including the Clostridia and related genera. Microbial AAA degradation commonly involves enzymes such as aminotransferases, dehydrogenases and decarboxylases and produces products including the corresponding aromatic metabolites such as arylpyruvate, arylpropionate, aryllactate, arylacrylate and arylacetate. The levels of phenylacetate, 3-phenylpropionate and 3-phenyllactate derived from phenylalanine catabolism, and 4-hydroxyphenyl lactate from tyrosine catabolism are elevated in intestinal diseases and sepsis Fedotcheva et al. Multiple enteric bacteria including E. coli, Proteus vulgaris, Paracolobactrum colifome , and Micrococcus aerogenes contain the enzyme tryptophanase EC 4. For example, Lactococci catabolize tryptophan via a tryptophan aminotransferase EC 2. Decarboxylases produce the corresponding primary aromatic amines from AAA Nakazawa et al. Phenylalanine is converted into phenylpyruvate by the action of an aryllactate dehydrogenase EC 1. Certain unusual reactions such as the cleavage of IPy into indole and pyruvate, the conversion of tyrosine to p -cresol via the decarboxylation of p -hydroxyphenylacetate and the formation of phenol from tyrosine via the elimination of ammonia and acetate, were also detected in intestinal anaerobic bacteria Smith and Macfarlane, The conversion of arylpyruvates into arylaldehydes is known; lactobacilli convert Ipy derived from tryptophan into indolealdehyde I3A and phenylalanine-derived phenylpyruvate into benzaldehyde Nierop Groot and de Bont, Perhaps the most distinctive pathway in all of AAA aerobic catabolism in the gut is found in several bacteria including E. coli and involves the degradation of phenylalanine via phenylacetate, through an unusual oxepin-CoA thioester synthesized by a multicomponent oxygenase, ultimately into acetyl-CoA and succinyl-CoA by means of β-oxidation Teufel et al. Fermentation of the AAA in the GI tract occurs in the strict anaerobes of the Firmicutes phylum. In Stickland fermentation, one AA donates electrons while another accepts them, thereby generating ATP and reducing power. Stickland electron donors include the branched chain AA, acidic AA and sulfur-containing AA as well as alanine, serine, histidine and phenylalanine; electron acceptors include glycine, proline, hydroxyproline, arginine, ornithine derived from arginine and tryptophan. The AAA can all be fermented as single AAs via the 2-hydroxyacid pathway, which has been studied extensively by Buckel and coworkers Kim et al. The pathway variant for AAA fermentation is termed the 3-aryllactate pathway and is discussed in detail in Radical dehydratases and the 3-aryllactate pathway. The end products of AAA reduction were known for some decades Elsden et al. However, all the intermediates involved in both AAA oxidation and reduction in Clostridium sporogenes were identified much later, and the enzymes responsible for the fermentation of phenylalanine Dickert et al. Initially, the issue of whether all the AAA were catabolized via the same radical dehydratase enzymes or different ones was unresolved Li, , but later work with mutants showed that only one dehydratase was active in the degradation of all the AAA via this pathway Dodd et al. The conversion of tryptophan to 3-indolepyruvate, oxidizing it to 3-indoleacetate, and reducing it via R indolelactate and E indoleacrylate to IPA, according to this pathway is depicted in Figure 5. Apart from C. sporogenes and C. botulinum Elsden et al. Interest in AAA catabolism in the gut had been earlier stimulated by the fact that one of the end products of tryptophan degradation, IPA, passes through the blood-brain barrier, scavenging reactive oxygen species ROS in the brain by the formation of kynuric acid Bendheim, , and thereby protecting it from Alzheimer's disease Chyan et al. Indeed, gut bacteria were shown to exert a large influence on the production of mammalian blood metabolites such as indoxyl sulfate and IPA; specifically IPA production was microflora-dependent and could be induced by the colonization of C. sporogenes Wikoff et al. IPA is also involved in strengthening gut-barrier function by directly acting on the pregnane X receptor PXR Venkatesh et al. Out of the 12 end products derived from the degradation of all the AAA via the reductive branch of this pathway, nine were detected recently in the host plasma Dodd et al. The same authors also proposed the genetic engineering of gut bacteria involved in producing major metabolites such as IPA, as a way to influence host health. Protozoa also occur frequently in ruminants and AAA catabolism by bacteria alone or bacteria in the presence of protozoa not only have differing utilization, but also different end products. For example, while rumen bacteria alone produced skatole, p -cresol and IPA as the end products, mixed bacterial-protozoan catabolism produced IAA, indolelactate and indole Mohammed et al. Tyrosine is converted by mixed cultures of bacteria and protozoa into p -hydroxyphenylacetic acid and further into p -cresol Mohammed et al. AAA catabolism also plays a role in bloodstream infections caused by protozoa. In Trypanosoma brucei , the conversion of phenylalanine into phenylpyruvate and tyrosine into p -hydroxyphenyllactate has been reported Stibbs and Seed, a. In the same organism, tryptophan was converted into indolelactate, IAA and tryptophol Stibbs and Seed, b. The existence of AAA-related dehydrogenases, transaminases and decarboxylases was surmised from these results. Since the levels of AAA catabolites shown to originate from the pathogen were elevated in infected animals, AAA catabolism might be important for the development of sleeping sickness Stibbs and Seed, c. An AAA-dehydrogenase or L-α-hydroxy acid dehydrogenase AHADH has been characterized in the causative agent of Chagas disease, T. cruzi , whereby phenyllactate and p -hydroxyphenyllactate were better substrates than indolelactate Montemartini et al. While the oxidation of AAA catabolic pathways in aerobes and anaerobes are often similar, the reductive branches of AAA fermentations are interesting due to the unique enzymes involved. The 3-aryllactate pathway is a type of 2-hydroxyacid pathway as mentioned earlier. Phenylalanine is fermented via 3-phenyllactate, tyrosine via 4-hydroxyphenyllactate and tryptophan via ILA, with all three fermentations involving catalysis by a common set of enzymes. In any 2-hydroxyacid pathway, the removal of non-acidic β-protons of 2-hydroxyacyl-CoA proceeds through a radical mechanism involving [4Fe-4S] clusters Buckel, Then, the arylpyruvate is oxidatively decarboxylated by a pathway-specific 2-ketoacid:ferredoxin-oxidoreductase with Coenzyme A CoA thioesterification to the corresponding acyl-CoA arylacetyl-CoA which is one carbon atom shorter than the arylpyruvate. Subsequently, substrate level phosphorylation SLP occurs with the formation of the oxidized end product, namely the arylacetate. In the reductive branch, the 2-ketoacid is reduced by NADH-dependent Re face -stereospecific dehydrogenase Berk et al. The resulting R hydroxyacid 3-aryllactate is thioesterified to the 2-hydroxyacyl-CoA 3-aryllactyl-CoA by specific CoA-transferases, but the β-protons still remain unactivated pKa ~ Therefore, a special enzyme of the 2-hydroxyacyl-CoA dehydratase 2-HADH family whose members share a common biochemical mechanism, the aryllactyl-CoA dehydratase, becomes necessary. In every 2-HADH, a substrate radical is generated via [4Fe-4S] clusters causes Umpolung charge reversal at the keto group of the R hydroxyacyl-CoA Buckel and Keese, The ketyl radical generated by one-electron reduction of the substrate eliminates water via proton transfer to a conserved glutamate residue Knauer et al. β-Proton abstraction by the Fe-O anion from one of the [4Fe-4S] clusters generates the allylic ketyl radical, which yields the E enoyl-CoA 3-arylacrylate in case of AAA degradation and recycles an electron. Hence, the reversible α,β- syn -dehydration of R hydroxyacyl-CoA into E enoyl-CoA is a key reaction, making 2-HADH a key enzyme Kim et al. Finally, 3-arylacrylate is reduced to the corresponding 3-arylpropionate. The reduced end product 3-arylpropionate has the same chain length as the respective parent AAA. In Clostridium sporogenes , the aryllactyl-CoA dehydratase complex also contains the CoA-transferase Dickert et al. One-electron reduction of a 2-HADH by its activator involves a large conformational change of the helix-[4Fe-4S] cluster-helix motif of the latter coupled to the hydrolysis of 2 ATP molecules Knauer et al. Once the electron transfer is complete, the activator dissociates from the 2-HADH, as deduced from chelation experiments Kim J. Therefore, electron transfer involving [4Fe-4S] clusters as the sole cofactors in the 2-HADH-activator system facilitates radical dehydrations with minimal ATP hydrolysis. The mechanism of the reaction is shown in Figure 6. Figure 6. Reaction mechanism of 3-aryllactyl-CoA dehydratase. The formation of the ketyl radical anion leads to the elimination of water via proton transfer to a conserved glutamate residue and the pK a of the beta-proton in the enoxy radcal is reduced from approximately 40 to about 15, a value which is further reduce by interactions with the active site residues. These highly negative redox potentials could be supplied by ferredoxin- or flavodoxin-oxidoreductases which oxidize arylpyruvates coupled to the addition of CoA, yielding arylacetyl-CoA. ATP formation is coupled to the reduction of 3-arylacrylates in the 3-aryllactate pathways of C. sporogenes Bader and Simon, Recent studies suggest that there is additional energy conservation in C. It can be seen from the preceding section that CoA-transferases are essential for AA degradation through the 2-hydroxyacid pathways. The genes encoding the CoA-transferases are located upstream of those encoding the corresponding 2-HADH. The aryllactate CoA-transferase FldA from C. sporogenes forms a complex with the aryllactyl-CoA dehydratase FldBC , which participates in the fermentation of AAA Dickert et al. FldA transfers the CoA moiety from arylacrylyl-CoA to aryllactate, after which FldBC catalyzes the radical dehydration. The arylacrylyl-CoA is then converted into acrylacrylate by FldA. The three enzymes FldA, HadA and CaiB were identified as belonging to a new family of proteins called the Type III CoA-transferases Heider, Unlike other CoA-transferases, catalysis by the Type III family entails a ternary complex mechanism without any intermediates covalently bound to the enzyme. Acid-R2 and the CoA-donor-R1 first bind non-covalently to the enzyme to form an anhydride whereby the released CoAthiolate stays at the enzyme. The attack of the CoA thiolate at the other acyl group of Acid-R2 forms the Acid-R1 and the new CoA-thioester, which are both released from the enzyme Heider, The presence of a CoA-ligase gene in many gene clusters containing Type III transferases indicates that catalytic amounts of CoA-thioesters are required to start the reaction and prevent depletion of the CoA-thioester pool by unspecific hydrolysis. The three AAA, their biosynthetic precursors, as well as modified non-protein AAA are important in the synthesis of a variety of antibiotics by bacteria and fungi. Phenylalanine is incorporated into the several antibiotics, for example the bacterial cell wall biosynthesis inhibiting mureidomycins Bugg et al. The biosynthesis of the polyketide enterocin contains a benzoyl-CoA precursor derived from the β-oxidation of trans -cinnamic acid, which in turn is synthesized from phenylalanine via PAL Piel et al. The biosynthesis of chlorobiocin also involves 3-dimethylallylhydroxybenzoic acid, which is derived from phenylalanine by prenylation and retro-aldol condensation Pojer et al. Tyrosine is the precursor for the biosynthesis of novobiocin, whose ring B is derived from the AAA via a coumarin intermediate Chen and Walsh, ; Pacholec et al. Examples of tryptophan-derived antibiotics include actinomycin, which later became well-known as a cancer drug Hollstein, Tryptophan-rich peptides such as indolicidin and tritrpticin, belong to a newer class of antimicrobial peptides Chan et al. Combining tryptophan residues with cationic AA like arginine generates antimicrobials able to penetrate bacterial cells effectively. Recently, researchers have developed lipopeptide analogs of polymyxin B often used in multi drug resistant cases that incorporate tryptophan Grau-Campistany et al. The shikimate pathway was believed to lead to the synthesis of the intermediate amino hydroxy benzoic acid AHBA , a compound which feeds into the biosynthesis of polyketide antibiotics of the ansamycin class, among which the anti-tubercular rifamycins are the most well-known Sensi et al. It was later discovered that the initial compounds of the shikimate pathway, PEP and E4P are converted in a few steps into AHBA via the aminoshikimate pathway, which contains steps similar to the shikimate pathway Ghisalba and Nüesch, ; Kim et al. Candicidin is an aromatic polyene macrolide antifungal molecule containing a 4-aminoacetophenone moiety Martin and McDaniel, ; Martin, , derived from chorismate via 4-aminobenzoic acid PABA by means of an aminotransferase reaction, with glutamine acting as the amino donor Gibson et al. Chorismate is also transformed into 4-hydroxybenzoic acid in bacteria by the action of chorismate lyase which belongs to the ubiquinone biosynthetic pathway Poon et al. Prephenate is the metabolic precursor of bacilysin produced by Bacillus subtilis , as elucidated by studies of mutants of phenylalanine and tyrosine biosynthesis Hilton et al. Dihydrophenylalanine, a non-protein AA and antibiotic produced by Photorhabdus luminescens , is generated via rerouting of prephenate by the action of an unusual non-aromatizing prephenate decarboxylase, followed by a transaminase Crawford et al. Anthranilate formed from tryptophan degradation; Figure 5 inhibits biofilm formation by Pseudomonas aeruginosa, Vibrio vulnificus, Bacillus subtilis, Salmonella enterica serovar Typhimurium, and Staphylococcus aureus , and disrupted biofilms already formed by these bacteria via multiple mechanisms Li et al. Therefore, anthranilate could potentially be used as a broad-spectrum biofilm inhibitor. AAA biosynthetic precursors as well as the AAA themselves are often rerouted into the production of non-canonical AAA analogs, which form parts of antibiotic scaffolds. Obafluorin, produced by Pseudomonas fluorescens , is biosynthesized via the key intermediate L-aminophenylalanine Herbert and Knaggs, Glycopeptide antibiotics such as the vancomycin and teicoplanin families, contain the non-canonical AAA analogs β-hydroxytyrosine β-Ht , 4-hydroxyphenylglycine Hpg and dihydroxyphenylglycine Dpg , all of which are capable forming rigid cross-links within the peptide. Among these, the biosynthesis of Dpg is not directly related to the shikimate pathway, but starts with the condensation of four malonyl-Coenzyme A molecules to 3,5-dihydroxyphenylacetyl-CoA DPA-CoA and three free coenzyme A CoASH Chen et al. The AAA biosynthesis intermediate prephenate is the starting point for the synthesis of Hpg, which involves the four enzymes described hence Hubbard et al. Prephenate dehydrogenase Pdh converts prephenate to p -hydroxyphenylpyruvate, followed by 4-hydroxymandelate synthase HmaS , which transforms p -hydroxyphenylpyruvate into L- p -hydroxymandelate and hydroxymandelate oxidase Hmo , which oxidizes L- p -hydroxymandelate to p -hydroxylbenzoylformate. Finally, transamination of the penultimate compound by p -hydroxyphenylglycine transaminase Pgat , yields Hpg. Enzymes involved in β-hydroxylation of non-ribosomal encoded amino acids were first characterized in organisms producing the antibiotics novobiocin and nikkomycin Chen and Walsh, ; Chen et al. Vancomycin biosynthesis involves similar enzymes. Tyrosine is first activated to a thiol ester and attached to one of the modular thioesterease enzyme domains for antibiotic synthesis. The thiol ester is oxidized by an oxygenase which adds a β-hydroxyl-group while the substrate is still attached to the thioesterase domain, and finally, Bht is released from the module Donadio et al. Chloro-β-hydroxytyrosine is also found in some glycopeptide antibiotics, but the chlorination time point was demonstrated to be later than the stage of free Bht synthesis Puk et al. Image resolution: High. Link: Normal Module. Background color Organism. ID search. Module Complete only Including 1 block missing Including any incomplete. |
| An Evolutionary Perspective on Amino Acids | LLC, Royal DSM, Showa Denko KK and Zhejiang Chemicals. Alternative substrates reveal catalytic cycle and key binding events in the reaction catalysed by anthranilate phosphoribosyltransferase from Mycobacterium tuberculosis. PS contributed to the experimental protocol, performed the experiments, data analysis, and wrote the manuscript. Through meticulous scrutiny of an array of illustrative case studies, we endeavor not only to unveil the intricate panorama of these modifications, but also to glean profound insights into their pivotal involvement in orchestrating the immune retort and intricacies of infection-associated metabolism. subtilis , B. Radkov AD, Moe LA Bacterial synthesis of d -amino acids. |
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