g , Schematic of RNA highlighting its ribose groups. h , Intracellular abundance of the four nucleoside precursors of RNA in control or UPP1 -FLAG-expressing K cells grown in sugar-free medium supplemented with 0.

with two-sided t -test relative to control. i , Cell growth assays of control or UPP1 -FLAG-expressing K cells in sugar-free medium supplemented with 0. Source data. To validate the screen, we stably expressed UPP1 and UPP2 ORFs in K cells and observed a significant gain in proliferation in galactose medium Fig.

The ability of UPP1 cells to grow in sugar-free medium strictly depended on uridine, and none of the other seven nucleoside precursors of nucleic acids could substitute for uridine Fig. Uridine-derived nucleotides are building blocks for RNA Fig.

We tested if RNA-derived uridine could support growth in a UPP1 -dependent manner and supplemented glucose-free medium with purified yeast RNA.

The intracellular abundance of all four ribonucleosides accumulated following addition of RNA to the medium, with significantly lower uridine levels in UPP1 -expressing cells, suggesting UPP1 -mediated catabolism Fig.

Accordingly, UPP1 -expressing K cells proliferated in sugar-free medium supplemented with RNA Fig. We conclude that elevated uridine phosphorylase activity confers the ability to grow in medium containing uridine or RNA, in the complete absence of glucose.

We next addressed the mechanism of how uridine supports the growth of UPP1 -expressing cells. Previous studies have noted the beneficial effect of uridine in the absence of glucose and proposed mechanisms that include the salvage of uridine for nucleotide synthesis and its role in glycosylation 4 , 5 , 6 , 7 , 8.

Others reported the beneficial role of uridine phosphorylase in maintaining ATP levels and viability during glucose restriction in the brain 9 , 10 , To further investigate the molecular mechanism of uridine-supported proliferation, we performed a secondary genome-wide CRISPR—Cas9 depletion screen using K cells expressing UPP1 -FLAG grown on glucose or uridine Fig.

and are corrected for natural isotope abundance. g , Schematic of uridine-derived ribose catabolism integrating gene essentiality results in glucose versus uridine. Gln, glutamine; Asp, aspartate. We found that, although most essential gene sets were shared between glucose and uridine conditions, three major classes of genes were differentially essential in uridine as compared to glucose Fig.

Accordingly, uridine-grown cells were particularly sensitive to depletion of PGM2 , TKT and RPE , or to TKT inhibition, while they were insensitive to the de novo pyrimidine synthesis inhibitor brequinar Fig. In contrast, genes of the oxidative branch of the PPP G6PD , PGLS , PGD did not score differentially between glucose and uridine.

Central enzymes of glycolysis were essential both in glucose and in uridine, indicating that a functional glycolytic pathway is required for survival with uridine alone. However, our comparative analysis revealed that several upper glycolytic enzymes encoded by ALDOA , GPI and HK2 were dispensable in uridine, and only essential in glucose Fig.

Not all steps of upper glycolysis scored in either condition, potentially due to the multiple genes with overlapping functions encoding glycolytic enzymes, a common limitation in single gene-targeting screens.

The essentiality of the non-oxPPP, with the dispensability of upper glycolysis in uridine Fig. Lactate secretion and glycolytic utilization of uridine, however, were excluded in earlier work 4 , 5 , 6 , 7 , 8. Nonetheless, given the importance of PPP enzymes and the dispensability of upper glycolysis, we reinvestigated this possibility and measured lactate secretion in uridine-grown cells.

Strikingly, we found that UPP1 -expressing cells grown in uridine secreted high amounts of lactate Fig. Accordingly, we found using liquid chromatography—mass spectrometry LC—MS that uridine restored steady-state abundance of most central carbon metabolism detected in the absence of glucose, strongly suggesting some degree of lower glycolysis activity from uridine Extended Data Fig.

To directly test if uridine-derived ribose could serve as a substrate for glycolysis, we designed a tracer experiment using isotopically labelled uridine with five ribose carbons 13 C 5 -uridine and LC—MS Fig.

UPP1 -expressing cells avidly incorporated 13 C 5 -uridine, as seen by the presence of 13 C in all the intracellular intermediates of the PPP and glycolysis analysed, including ribose-phosphate, upper and lower glycolytic intermediates and lactate, while control cells showed very little label incorporation.

As in cell lines, we found 13 C incorporation in ribose-phosphate and glycolysis in 13 C 5 -uridine-treated animals Fig. Incorporation efficiency was smaller than in cell culture, as expected from low-dose 13 C 5 -uridine injection, shorter treatment time and competition with other endogenous substrates in vivo, including unlabelled uridine.

We also found modest but significant incorporation of uridine-derived 13 C in glucose, indicating gluconeogenesis from uridine-derived carbons Fig. Together, our results indicate that in cell lines and in animals in vivo, uridine catabolism provides ribose for the PPP, and that the non-oxPPP and the glycolytic pathway communicate via F6P and G3P to replenish glycolysis thus entirely bypassing the requirement for glucose in supporting lower glycolysis, biosynthesis and energy production in sugar-free medium Fig.

We next sought to determine whether any human cell lines exhibit a latent ability to use uridine-derived ribose to grow on uridine when glucose is absent without the need for over-expression.

Cells from the melanoma and the glioma lineages grew remarkably well in uridine as compared to the other lineages, whereas Ewing sarcoma cells grew significantly less well Fig. Cell lines from the PRISM collection have been extensively characterized at a molecular level 14 , so we searched for genomic factors that correlate with the ability to grow on uridine Supplementary Table 1.

Genome wide, the top-scoring transcript, protein and genomic copy number variant was UPP1 Fig. Expression of UPP1 across the CCLE collection was the highest in cell lines of skin origin Extended Data Fig.

In agreement with these results, we confirmed significant, UPP1 -dependent, proliferation and uridine catabolism in melanoma cells grown in sugar-free medium supplemented with uridine or RNA Fig.

We conclude that the endogenous expression of UPP1 is necessary and sufficient to support the growth of cancer cells on uridine. False discovery rates FDRs were calculated using a Benjamini—Hochberg algorithm correcting for multiple comparisons UPP1 is encoded on Chr7p MDA, MDA-MBS.

with two-sided t -test relative to untreated cells. We next investigated the factors that promote UPP1 expression and growth on uridine by integrating our results with CCLE data to prioritize transcription factors, which highlighted MITF as a strong candidate in melanoma cells, both at the protein and the transcript level Fig.

We found that MITF over-expression promoted UPP1 expression and uridine growth Extended Data Fig. Accordingly, siRNA-mediated depletion of MITF decreased UPP1 expression in melanoma cells Extended Data Fig. Our solid tumour PRISM cancer cells collection did not include cells of the immune lineage, where UPP1 is expressed at high levels 17 , 18 , so we asked whether immune cells exhibit the capacity to metabolize ribose from uridine either at baseline or in a transcriptionally regulated manner.

Among the immunostimulatory molecules, RNA enhanced UPP1 expression, suggesting the existence of a feed-forward loop, where RNA and conceivably RNA-containing pathogens and debris may trigger UPP1 expression and uridine salvage for building blocks and energy production.

Label incorporation from uridine ribose was also strongly increased in citrate and lactate after differentiation of THP1 and after BMDM stimulation with R, while it wasn't further increased in M-CSF-matured PBMCs, possibly due to high baseline capacity for uridine catabolism in these cells Fig.

Together, our results indicate that macrophages have the capacity to use uridine-derived ribose for glycolysis, and that UPP1 expression and uridine catabolism can sharply increase during cellular differentiation and in response to immunostimulating molecules, with cell type and species differences.

We next sought to determine whether glycolysis from uridine is under acute regulation in the same way as from glucose. Active OXPHOS tends to keep glucose uptake and glycolysis at lower levels, while acute inhibition of OXPHOS leads to an immediate and strong increase in glucose-supported glycolysis, as evidenced by a robust increase in the extracellular acidification rate ECAR following oligomycin treatment Fig.

Strikingly, we found no ECAR stimulation by OXPHOS inhibitors, no difference in 13 C 5 -uridine incorporation following antimycin blockage of the electron transport chain, and no increase in uridine import in OXPHOS-inhibited UPP1 -expressing cells grown on uridine Fig.

Because glycolysis from both uridine and glucose share a common pathway from G3P Fig. Consistent with this notion, we observed no stimulation of ECAR in mannose-grown cells, a sugar connected to glycolysis by F6P Extended Data Fig.

We conclude that substrates such as uridine can enter glycolysis in a constitutive way, in contrast to glucose, by bypassing regulatory steps of upper glycolysis such as glucose transport and initial phosphorylation. a , Schematic of glycolysis inhibition by OXPHOS. G6P, glucosephosphate. O, oligomycin; C, CCCP; A, antimycin A.

In line with this, we next performed a competition experiment to evaluate if the presence of glucose affects the incorporation of uridine in cells. Incorporation of uridine in lactate was notably not affected by competition with glucose in our experimental conditions, despite the presence of a large molar excess of glucose Fig.

Therefore, and in agreement with a bypass of regulatory steps of upper glycolysis, uridine can be incorporated into cells even when lactate production from glucose is saturated, suggesting constitutive import and catabolism. Cells with severe OXPHOS dysfunction classically have to be grown on glucose, and uridine must be supplemented 1.

The traditional explanation has been that glucose is required to support glycolytic ATP production as OXPHOS is debilitated, and that uridine supplementation is required for pyrimidine salvage given that de novo pyrimidine synthesis via DHODH requires coupling to a functional electron transport chain 1 , 3 Extended Data Fig.

Having observed energy harvesting from uridine, we finally tested whether uridine-derived ribose could also benefit OXPHOS-inhibited cells in the absence of glucose. We found a significant UPP1 -dependent rescue of viability in galactose-grown cells treated with antimycin A Fig.

For decades it has been known that cells with mitochondrial deficiencies are dependent on uridine to support pyrimidine synthesis given the dependence of de novo pyrimidine synthesis on DHODH, whose activity is coupled to the electron transport chain 1. Although it has been documented, it is less appreciated that uridine supplementation can support cell growth in the absence of glucose 4 , 5 , 6 , 7 , 8 , 9 , Here, we show that, in addition to nucleotide synthesis, uridine can serve as a substrate for energy production, biosynthesis and gluconeogenesis.

By comparing uridine to other nucleosides and using similar tracer experiments to ours, Wice et al. However, they did not detect pyruvate and lactate in uridine, and concluded that uridine does not participate in glycolysis, but rather is required for nucleotide synthesis, and proposed that energy is derived exclusively from glutamine in the absence of glucose 6 , 7.

Loffler et al. and Linker et al. reached the same conclusion 4 , 8. Our observations based on a genome-wide CRISPR—Cas9 screening and metabolic tracers Fig. It has previously been reported that uridine protects cortical neurons and immunostimulated astrocytes from glucose deprivation-induced cell death, in a way related to ATP, and it was hypothesized that uridine could serve as an ATP source 9.

Our genetic perturbation and tracer studies are consistent with this hypothesis. The capacity to harvest energy and building blocks from uridine appears to be widespread. Here, we report very high capacity for uridine-derived ribose catabolism in melanoma and glioma cell lines Fig.

Based on gene expression atlases 18 , 19 , we predict uridine may be a meaningful source of energy in blood cells, lung, brain and kidney, as well as in certain cancers. Uridine is the most abundant and soluble nucleoside in circulation 20 and it is possible that uridine may serve as an alternative energy source in these tissues, or for immune and cancer metabolism, similar to what has been proposed for other sugars and nucleosides 21 , 22 , It is notable that the strongest human metabolic quantitative trait loci for circulating uridine corresponds to UPP1 ref.

A fascinating aspect of glycolysis from uridine is its apparent absence of regulation, at least at shorter timescales. The ability of uridine to serve as a constitutive input into glycolysis might have clinical implications for human diseases, as uridine is present at high levels in foods such as milk and beer 26 , 27 , and previous in vivo studies have shown that a uridine-rich diet leads to glycogen accumulation, gluconeogenesis, fatty liver and pre-diabetes in mice 28 , We now report that glycolysis from uridine lacks at least two checkpoints as 1 it is not controlled by OXPHOS Fig.

Although glycolysis from uridine appears to occur at a slower pace than from glucose, we speculate that constitutive fuelling of glycolysis and gluconeogenesis from a uridine-rich diet may contribute to human conditions such as fatty liver disease and diabetes.

This ability of uridine to bypass upper glycolysis may be beneficial in certain cases. At longer timescales, UPP1 expression and capacity for ribose catabolism from uridine appear to be determined by cellular differentiation and further activation by extracellular signals.

Here we focused on the monocytic lineage and found that 1 in THP1 cells, UPP1 expression and activity sharply increased during differentiation and polarization, 2 high baseline rates of glycolysis from uridine are observed in M-CSF-matured PBMCs and 3 treatment with immunostimulating molecules acutely promote both UPP1 expression and uridine catabolism in BMDMs Fig.

It is thus likely that NF-κB may serve as a transcription factor for UPP1. Supporting this assertion, we found that blocking NF-κB signalling with upstream IKK inhibitors abolished Rinduced Upp1 expression Extended Data Fig. Uridine phosphorylase and ribose salvage by UPP1 appears to lie downstream of a number of signalling pathways with potential relevance to disease.

We have demonstrated that uridine breakdown is promoted by MITF, a transcription factor associated with melanoma progression, which we show binds upstream of UPP1 to promote its expression Extended Data Fig. In an accompanying study, Nwosu, Ward et al. It is notable that both MITF and NF-kB can act downstream of KRAS—MAPK 34 , 35 , 36 , 37 , 38 and that some pancreatic cell lines with high uridine phosphorylase activity highlighted by Nwosu, Ward et al.

Finally, we found that RNA in the medium can replace glucose to promote cellular proliferation Fig. Recycling of ribosomes through ribophagy, for example, plays an important role in supporting viability during starvation Cells of our immune system also ingest large quantities of RNA during phagocytosis, and we experimentally showed that the expression of UPP1 increases with macrophage activation Fig.

Uridine seems to be the only constituent of RNA that can be efficiently used for energy production, at least in K cells Fig. Whereas the salvage of RNA to provide building blocks during starvation has long been appreciated for nucleotide synthesis, to our knowledge, its contribution to energy metabolism has not been considered in the past, except for some fungi that can grow on minimum media with RNA as their sole carbon source We speculate that, similar to glycogen and starch, RNA itself may constitute as large stock of energy in the form of a polymer, and that it may be used for energy storage and to support cellular function during starvation, or during processes associated with high energy costs such as the immune response.

K CCL , T CRL , HeLa CCL-2 , A CRL , A CRL , SH4 CRL , MDA-MBS HTB , SK-MEL-5 HTB , SK-MEL HTB and THP1 TIB cell lines were obtained from the American Type Culture Collection ATCC.

UACC, UACC and LOX-IMVI cells were obtained from the Frederick Cancer Division of Cancer Treatment and Diagnosis DCTD Tumor Cell Line Repository.

All cell lines were re-authenticated by STR profiling at ATCC before submission of the manuscript and compared to ATCC and Cellosaurus ExPASy STR profiles in , with the exception of THP1 TIB and U CRL Cells lines from the PRISM collection were obtained from The PRISM Lab Broad Institute and were not further re-authenticated.

MDA-MBS cells were previously assumed to be ductal carcinoma cells and recent gene expression analysis assigned them to the melanoma lineage ATCC. All growth assays, metabolomics, screens and bioenergetics experiments were performed in medium containing dialysed FBS.

For RNA and other nucleoside complementation assays, 0. Cell viability in glucose and galactose was determined using the same Vi-Cell Counter assay. Measurements were taken from distinct samples. For ORF screening, K cells were infected with a lentiviral-carried ORFeome v8. Cells were infected at a multiplicity of infection of 0.

Barcode sequencing, mapping and read count were performed by the Genome Perturbation Platform Broad Institute. For screen analysis, log 2 normalized read counts were used, and P values were calculated using a two-sided t -test.

The presence of lentiviral recombination within the ORFeome library was not tested and as such genes that dropped out should be considered with caution, as these may represent unnatural proteins Twenty-four hours after infection, cells were selected with 0.

Protein concentration was determined from total cell lysates using a DC protein assay Bio-Rad. Gel electrophoresis was done on Novex Tris-Glycine gels Thermo Fisher Scientific before transfer using the Trans-Blot Turbo blotting system and nitrocellulose membranes Bio-Rad. Washes were done in TBST.

Specific primary antibodies were diluted at a concentration of —, in blocking buffer. Fluorescent-coupled secondary antibodies were diluted at a ratio of , in blocking buffer.

Membranes were imaged with an Odyssey CLx analyzer Li-cor with Image Studio Lite v4. The following antibodies were used: FLAG M2 Sigma, F , Actin Abcam, ab , TUBB Thermo, MA , UPP1 Sigma, SAB , MITF Sigma, HPA , TYR Santa Cruz sc , MLANA CST, , HK2 CST, , GPI CST, , ALDOA CST, , TKT CST, , RPE Proteintech, AP , PGM2 Proteintech, AP , UCK2 Proteintech, AP , TYMS Proteintech, AP , S6 ribosomal protein Santa Cruz, sc and phosphor-S6 Santa Cruz, sc Two commercially available antibodies to UPP2 were tested Sigma, SAB; Abcam, ab , but no specific band could be detected.

The medium was replaced with fresh medium on days 3 and 5. On day 6, all wells reached confluency and cells were lysed. Barcode abundance was determined from sequencing, and unexpectedly low counts for example, from sequencing noise were filtered out from individual replicates so as not to unintentionally depress cell line counts in the collapsed data.

Replicates were then mean-collapsed, and log fold change and growth rate metrics were calculated according to equations 1 and 2 :. where n u and n g are counts from the uridine and glucose supplemented conditions, respectively, n 0 and n f are counts from the initial and final timepoints, respectively, and t is the assay length in days.

Data analysis and correlation analysis were performed by The PRISM Lab following a published workflow qPCR was performed using the TaqMan assays Thermo Fisher Scientific. Human PBMCs and mouse BMDM data were normalized to TBP , and liver mouse data were normalized to Rplp2 , both using the ΔΔCt method.

qPCR primers for ChIP are described below. Fixation was stopped by adding glycine final concentration of 0. Cells were harvested by scraping with ice-cold PBS. Samples were centrifuged to remove debris and diluted tenfold in immunoprecipitation dilution buffer DNA was purified with QIAquick PCR purification kit Qiagen.

Purified DNA was co-transfected with a GFP-expressing plasmid in the cell lines of interest using Lipofectamine Thermo Fisher Scientific. UPP1 depletion in single-cell clones was assessed by protein immunoblotting using antibodies to UPP1.

The 9-bp deletion in clone 2 is expected to produce a truncated protein hypomorphic allele. Three hours after plating, cells were further treated with 0. Human PBMCs were isolated from buffy coats of blood donors from a local transfusion centre.

On day 6, cells were detached, counted and replated at 1. PBMC polarization was performed as with BMDMs. A secondary genome-wide CRISPR—Cas9 screening was performed using K cells expressing UPP1 -FLAG and a lentiviral-carried Brunello library Genome Perturbation Platform, Broad Institute containing 76, sgRNAs 44 , in duplicate.

Cells were infected with multiplicity of infection of 0. DNA isolation was performed as for the ORFeome screen. The log 2 fold change of each sgRNA was determined relative to the pre-swap control. For each gene in each replicate, the mean log 2 fold change in the abundance of all four sgRNAs was calculated.

log 2 fold changes were averaged by taking the mean across replicates. For each treatment, a null distribution was defined by the 3, genes with lowest expression.

To score each gene within each treatment, its mean log 2 fold change across replicates was z -score transformed, using the statistics of the null distribution defined as above. Cells were incubated for five additional hours before metabolite extraction.

All animal experiments in this paper were approved by the Massachusetts General Hospital, the University of Massachusetts Institutional Animal Care and Use Committee, or the Swiss Cantonal authorities, and all relevant ethical regulations were followed. All cages were provided with food and water ad libitum.

Food and water were monitored daily and replenished as needed, and cages were changed weekly. A standard light—dark cycle of h light exposure was used. Animals were housed at 2—5 per cage. Liver was flash frozen in liquid nitrogen before subsequent analysis, and blood was collected in EDTA plasma tubes, spun and plasma was stored for further analysis.

For each run, the total flow rate was 0. Data were acquired using Xcalibur v. Data were analysed using TraceFinder v. The flow rate was then increased to 0. Approximately 1. FBS was omitted.

Data were analysed using the Seahorse Wave Desktop Software v. Data were not corrected for carbonic acid derived from respiratory CO 2. Lactate secretion in the culture medium was determined using a glycolysis cell-based assay kit Cayman Chemical.

Cells were then re-counted and seeded in fresh medium of the same formulation and incubated for three additional hours. Cells were then spun down and lactate concentration was determined on the supernatants spent media.

Gene Ontology GO analysis was performed using GOrilla with default settings and using a ranked gene list as input The complete unfiltered data can be found in Supplementary Table 1. cDNAs of interest were custom designed Genewiz or IDT and cloned into pWPI-Neo or pLV-lenti-puro using BamHI and SpeI New England Biolabs.

All reported sample sizes n represent biological replicate plates or a different mouse. All attempts at replication were successful.

Statistical tests were performed using Microsoft Excel and GraphPad Prism 9. Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article. All data generated or analysed during this study are included in the article and its Supplementary Information.

Results of the ORFeome, the CRISPR—Cas9 and the PRISM screens are available in Supplementary Table 1. Source data are provided with this paper. King, M. Human cells lacking mtDNA: repopulation with exogenous mitochondria by complementation.

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Urasaki, Y. Chronic uridine administration induces fatty liver and pre-diabetic conditions in mice. Without sufficient energy, cells cannot maintain integrity and function. Supplemental D-ribose has been shown to improve cellular processes when there is mitochondrial dysfunction.

When individuals take supplemental D-ribose, it can bypass part of the pentose pathway to produce D-ribosephosphate for the production of energy.

In this article, we review how energy is produced by cellular respiration, the pentose pathway, and the use of supplemental D-ribose.

Keywords: Adenosine Triphosphate; Bioenergetics; D-ribose; Mitochondria.

Mitochondria are important organelles referred to as cellular powerhouses for their unique properties of cellular energy production. With many pathologic conditions and aging, mitochondrial function declines, and there is a reduction in the production of adenosine triphosphate.

The energy carrying molecule generated by cellular respiration and by pentose phosphate pathway, an alternative pathway of glucose metabolism. Published May 1, - Version history. Browse pages Click on an image below to see the page.

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Go to Top Version history. Sign up for email alerts. Among the 25 species with Rubisco, 14 harbor an NMP phosphorylase homolog, while 11 do not. It thus seems that there are three major variations of the pentose bisphosphate pathway in halophiles that account for 42 of the 63 species shown in Table 1 ; i one with Rubisco and NMP phosphorylase, as seen in members of Thermococcales Fig.

The distribution of these variations among the halophilic archaea is not linked to the phylogenetic relationships of their source organisms Fig. This raises the possibility that there may be even more variations of nucleoside degradation pathways in halophilic archaea that involve pentose bisphosphates.

The results obtained in this study and the distribution of relevant genes imply the occurrence of three types of nucleoside degradation pathways in halophilic archaea.

The three metabolic routes are with NMP phosphorylase and Rubisco a , with Rubisco but without NMP phosphorylase b , without Rubisco or NMP phosphorylase but with RuBP phosphatase and Ru1P aldolase c. The phylogenetic tree was constructed using 16S rRNA gene sequences.

A sequence from Thermoplasma volcanium tvo was used as an outgroup. The colors of the circles correspond to those of the nucleoside metabolic pathways in Fig. Organisms shown in red and pink codes indicate the halophilic archaea shown in Fig.

The organisms in red codes show those whose proteins were actually examined in this study. Among them, Halorhabdus utahensis and Halorhabdus tiamatea harbor homologs of the classical NOPP pathway found in bacteria and eukaryotes, and the NOPP pathway may be responsible for nucleoside degradation in these species.

However, there are still 19 halophilic archaea whose genome sequences do not provide any clues as to how they carry out nucleoside degradation. The homolog tends to occur in these halophiles, including 9 species in Haloarcula , Haloplanus , and Halohasta , and might provide clues to identify additional pathways involved in nucleoside or pentose metabolism.

Analysis of recombinant Hl -Urdpase1 indicated that the enzyme preferred guanosine as the nucleoside substrate for the phosphorylase reaction. In addition to the guanosine phosphorylase, almost all halophilic archaea harbor a second uridine phosphorylase homolog, Urdpase2.

Although these second homologs do not form an operon with genes of the pentose bisphosphate pathway, there is a high possibility that the gene products also generate R1P via nucleoside phosphorolysis.

Phylogenetic analysis clearly revealed that Urdpase1 and Urdpase2 are distinct Supplementary Fig. Identifying the nucleoside substrate of Urdpase2 will add to our understanding of nucleoside degradation in halophilic archaea.

It is intriguing that only one of the two nucleoside phosphorylase genes, the guanosine phosphorylase gene, forms an operon with the genes of the non-carboxylating pentose bisphosphate pathway.

This may be related to the high GC contents in genomes of halophilic archaea such as H. salinarum Two R1P kinases have been identified in previous studies; an ADP-dependent R1P kinase from the hyperthermophilic archaeon T.

When comparing the properties of the three enzymes, the R1P kinase from T. kodakarensis is ADP-dependent, whereas the enzymes from P. calidifontis and H. salinarum are ATP-dependent. On the other hand, the enzymes from T.

kodakarensis and H. xanaduensis display strict substrate specificity toward R1P, while the R1P kinase from P. calidifontis can recognize cytidine and uridine in addition to R1P Neither R15P isomerase nor NMP phosphorylase gene homologs are present in P.

calidifontis , resembling the case of the ten halophilic archaea with only nucleoside phosphorylase and ATP-R1PK homologs described above. On the other hand, the presence of R1P kinase in E. coli has been suggested A protein encoded by phnN was identified that displays kinase activity towards R15P, leading to the production of PRPP.

A protein responsible for the proposed second reaction, although unidentified, would be an R1P kinase. coli possesses several enzymes displaying similarity with the archaeal proteins that display R1P kinase activity.

As these include proteins whose functions have not been determined, one of them may also be R1P kinase. R1P kinase and its product, R15P, may be distributed through Archaea and Bacteria more widely than previously expected.

Unless mentioned otherwise, chemical reagents were purchased from Nacalai Tesque Kyoto, Japan , FUJIFILM Wako Pure Chemicals Osaka, Japan , or Merck Darmstadt, Germany.

Strains and plasmids used in this study are listed in Supplementary Table 2. When necessary, nucleosides were added into the medium. Escherichia coli DH5α strain used for plasmid construction and E. Plasmids to express genes encoding Hs -R15P isomerase and Hs -Urdpase1 were constructed as follows.

Coding regions of Hs -R15P isomerase and Hs -Urdpase1 genes were amplified by PCR using genomic DNA from H. Sequences of these primers are listed in Supplementary Table 3. The resulting expression plasmids are designated pET-Hs-R15P isomerase and pET-Hs-Urdpase1 Supplementary Table 2.

Expression plasmids for genes encoding Ht -R15P isomerase, Hs -RbsK, Ht -RbsK, Hx -RbsK, Ht -Urdpase1, Hx -Urdpase1, Hl -Urdpase1, Hx -HAD hydrolase, Hx -FucA, and Hs -GaR Supplementary Table 2 were prepared as follows.

Genes were designed and synthesized Integrated DNA Technologies, Coralville, IA to decrease their GC contents, and optimize their codons to enhance gene expression in E. For all 10 resulting expression plasmids Supplementary Table 2 with the two plasmids described above, we carried out DNA sequencing analysis and confirmed the absence of unintended mutation.

The designed sequences of each gene are shown in Supplementary Fig. The constructed expression plasmids were introduced into E. coli Rosetta DE3 for genes encoding R15P isomerase, RbsK, HAD hydrolase, and FucA or E. coli BLCodonPlus DE3 -RIL for genes encoding Urdpase1 and GaR.

Proteins were eluted with a linear gradient of NaCl 0 to 1. The same buffer was used as mobile phase. Hl -Urdpase1 and Hx -HAD hydrolase recombinant proteins were purified with Bio-Scale CHT and Superdex After centrifugation, the supernatants were applied to Bio-Scale CHT equilibrated with the same buffers used for cell suspension.

The concentrations of the purified enzymes were determined with the Protein Assay System Bio-Rad , using bovine serum albumin BSA Thermo Fisher Scientific, Waltham, MA as a standard. Protein homogeneity was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE.

After cell density OD reached 0. Fractions displaying glycolaldehyde reductase activity were collected. The same buffer was used as a mobile phase. Protein concentration was determined as described above. The amino acid sequence of the purified protein exhibiting glycolaldehyde reductase activity was identified by LC-MS analysis.

After separation with SDS-PAGE, proteins were stained with silver staining. The portion of the gel containing the target protein was excised and destained with Silver Stain MS Kit.

The protein in the gel was reduced, alkylated, digested with trypsin, and extracted using In-Gel Tryptic Digestion Kit Thermo Fisher Scientific. The trypsin-digested peptides were analyzed by nano-flow reverse phase liquid chromatography followed by tandem MS, using an LTQ-Orbitrap XL hybrid mass spectrometer Thermo Fisher Scientific.

After washing the trap with MS-grade water containing 0. Xcalibur 2. Fragmentation was performed by collision induced dissociation CID. Gene searches were carried out by using the SEQUEST-HT Thermo Fisher Scientific.

R15P isomerase activity was examined using ribose-1,5-bisphosphate R15P as the substrate and detecting the product RuBP with HPLC. The eluted compounds were monitored with a differential refractive index detector.

HPLC chromatogram data measured with LCA or Nexera X2 systems Shimadzu, Kyoto, Japan were collected using LCsolution 1. PK converts phosphoenolpyruvate and NDPs into pyruvate and NTPs. To measure activity of Hx -RbsK, NDP production was examined as follows. Eighty-five phosphate acceptors tested as substrates and reaction conditions are summarized in Supplementary Table 1.

The coupling enzymes were added and A was monitored at room temperature until a decrease was no longer observed. Unless described otherwise, absorbance was measured with a spectrophotometer, Ultrospec GE Healthcare or Ultrospec pro GE Healthcare.

The reaction without phosphate acceptor substrate was carried out and the value was subtracted from the values obtained with the acceptor substrates. The amounts of produced NDPs were normalized by dividing with reaction time min and the amount of protein mg.

RuBP formation was detected by HPLC as described above. Nucleoside phosphorylase activity of Hl -Urdpase1 was measured by quantifying the released nucleobase with HPLC. The reaction products were analyzed by HPLC using a COSMOSIL 5CPAQ packed column 4.

Adenosine, adenine, inosine, hypoxanthine, guanosine, guanine, uridine, uracil, cytidine, cytosine, thymidine, and thymine were utilized as standard compounds to prepare standard curves. In all kinetic analyses of enzymes, curve fitting and calculation of V max and K m values were carried out with IGOR Pro version 6.

Phosphatase activity of Hx -HAD hydrolase was determined by quantifying released phosphate with a malachite green assay. Twelve substrates, glucosephosphate G1P , glucosephosphate G6P , glucose-1,6-bisphosphate G16P , fructosephosphate F1P , fructosephosphate F6P , fructose-1,6-bisphosphate F16P , ribosephosphate R5P , ribosephosphate R1P , ribose-1,5-bisphosphate R15P , ribulosephosphate Ru5P , ribulose-1,5-bisphosphate RuBP , and p -nitrophenylphosphate pNPP , were examined.

Released free phosphate was quantified using Malachite Green Phosphate Assay kits BioAssay Systems, Hayward, CA. Aldolase activities of Hx -FucA were measured with dihydroxyacetone phosphate DHAP and various aldehydes as substrates. Residual DHAP after the condensation reaction was quantified with glycerolphosphate dehydrogenase GPDH by measuring NADH consumption.

After the addition of GPDH, A was monitored with an Ultrospec pro until a decrease was no longer observed and residual DHAP was quantified based on the amount of consumed NADH. The Hx -HAD hydrolase reaction coupled with the Hx -FucA reaction was examined by detecting DHAP production from RuBP.

DHAP production was quantified as described above. Kinetic parameters of Hx -FucA protein toward DHAP and glycolaldehyde were determined as follows. As Ru1P is not commercially available, the Hx -HAD hydrolase protein was utilized to produce Ru1P for constructing a standard curve of Ru1P.

The reaction was carried out as follows. Reductase activities toward glycolaldehyde and DHAP were measured by monitoring NADH consumption A in the reaction mixture with a UV spectrophotometer, UV Shimadzu and data was collected with UVProbe 2.

Data analysis was carried out with UVProbe 2. Oxidase activity toward sn -glycerolphosphate was examined as follows. The reaction product of Hx -HAD hydrolase was analyzed by NMR. The reaction product was concentrated three times by vacuum drying, appropriately diluted with D 2 O D, The 1 H-NMR spectra were acquired using a pulse sequence incorporating presaturation for water suppression.

The chemical shifts of the 1 H-NMR spectra were given in ppm relative to the signals of solvents using external standards of D 2 O at 4. The obtained NMR data was analyzed with Alice2 version 6. When measuring guanosine phosphorylase activity, cells were cultured without nucleosides to decrease background signals deriving from the added nucleosides.

Cell-free extracts were prepared as described for purifying GaR. The products R1P, R15P, RuBP, Ru1P, DHAP, and ethylene glycol were monitored by a differential refractive index detector. Sequences of 16S ribosomal RNA genes from halophiles were obtained from the KEGG Genes database.

When there were multiple 16S ribosomal RNAs in one organism, one sequence was randomly selected. Further information on research design is available in the Nature Research Reporting Summary linked to this article. Uncropped gel images of the gel images shown in Supplementary Figs.

All data for bar graphs in Fig. Artificial gene sequences encoding Ht -R15P isomerase, Hs -RbsK, Ht -RbsK, Hx -RbsK, Ht -Urdpase1, Hx -Urdpase1, Hl -Urdpase1, Hx -HAD hydrolase, Hx -FucA, and Hs -GaR are deposited in GenBank with the accession numbers LC, LC, LC, LC, LC, LC, LC, LC, LC, and LC, respectively.

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