Serine Hydroxymethyltransferase Activity Research Articles

Vitamin B6 deficiency cooperates with oncogenic Ras to induce malignant tumors in Drosophila

Vitamin B6 is a water-soluble vitamin which possesses antioxidant properties. Its catalytically active form, pyridoxal 5’-phosphate (PLP), is a crucial cofactor for DNA and amino acid metabolism. The inverse correlation between vitamin B6 and cancer risk has been observed in several studies, although dietary vitamin B6 intake sometimes failed to confirm this association. However, the molecular link between vitamin B6 and cancer remains elusive. Previous work has shown that vitamin B6 deficiency causes chromosome aberrations (CABs) in Drosophila and human cells, suggesting that genome instability may correlate the lack of this vitamin to cancer. Here we provide evidence in support of this hypothesis. Firstly, we show that PLP deficiency, induced by the PLP antagonists 4-deoxypyridoxine (4DP) or ginkgotoxin (GT), promoted tumorigenesis in eye larval discs transforming benign RasV12 tumors into aggressive forms. In contrast, PLP supplementation reduced the development of tumors. We also show that low PLP levels, induced by 4DP or by silencing the sgllPNPO gene involved in PLP biosynthesis, worsened the tumor phenotype in another Drosophila cancer model generated by concomitantly activating RasV12 and downregulating Discs-large (Dlg) gene. Moreover, we found that RasV12 eye discs from larvae reared on 4DP displayed CABs, reactive oxygen species (ROS) and low catalytic activity of serine hydroxymethyltransferase (SHMT), a PLP-dependent enzyme involved in thymidylate (dTMP) biosynthesis, in turn required for DNA replication and repair. Feeding RasV12 4DP-fed larvae with PLP or ascorbic acid (AA) plus dTMP, rescued both CABs and tumors. The same effect was produced by overexpressing catalase in RasV12DlgRNAi 4DP-fed larvae, thus allowing to establish a relationship between PLP deficiency, CABs, and cancer. Overall, our data provide the first in vivo demonstration that PLP deficiency can impact on cancer by increasing genome instability, which is in turn mediated by ROS and reduced dTMP levels.

One substrate many enzymes virtual screening uncovers missing genes of carnitine biosynthesis in human and mouse

The increasing availability of experimental and computational protein structures entices their use for function prediction. Here we develop an automated procedure to identify enzymes involved in metabolic reactions by assessing substrate conformations docked to a library of protein structures. By screening AlphaFold-modeled vitamin B6-dependent enzymes, we find that a metric based on catalytically favorable conformations at the enzyme active site performs best (AUROC Score=0.84) in identifying genes associated with known reactions. Applying this procedure, we identify the mammalian gene encoding hydroxytrimethyllysine aldolase (HTMLA), the second enzyme of carnitine biosynthesis. Upon experimental validation, we find that the top-ranked candidates, serine hydroxymethyl transferase (SHMT) 1 and 2, catalyze the HTMLA reaction. However, a mouse protein absent in humans (threonine aldolase; Tha1) catalyzes the reaction more efficiently. Tha1 did not rank highest based on the AlphaFold model, but its rank improved to second place using the experimental crystal structure we determined at 2.26 Å resolution. Our findings suggest that humans have lost a gene involved in carnitine biosynthesis, with HTMLA activity of SHMT partially compensating for its function.

Novel tetrahydrofolate-dependent d-serine dehydratase activity of serine hydroxymethyltransferases.

d-Serine plays vital physiological roles in the functional regulation of the mammalian brain, where it is produced from l-serine by serine racemase and degraded by d-amino acid oxidase. In the present study, we identified a new d-serine metabolizing activity of serine hydroxymethyltransferase (SHMT) in bacteria as well as mammals. SHMT is known to catalyze the conversion of l-serine and tetrahydrofolate (THF) to glycine and 5,10-methylenetetrahydrofolate, respectively. In addition, we found that human and Escherichia coli SHMTs have d-serine dehydratase activity, which degrades d-serine to pyruvate and ammonia. We characterized this enzymatic activity along with canonical SHMT activity. Intriguingly, SHMT required THF to catalyze d-serine dehydration and did not exhibit dehydratase activity toward l-serine. Furthermore, SHMT did not use d-serine as a substrate in the canonical hydroxymethyltransferase reaction. The d-serine dehydratase activities of two isozymes of human SHMT were inhibited in the presence of a high concentration of THF, whereas thatof E. coli SHMT was increased. The pH and temperature profiles of d-serine dehydratase and serine hydroxymethyltransferase activities of these three SHMTs were partially distinct. The catalytic efficiency (kcat /Km ) of dehydratase activity was lower than that of hydroxymethyltransferase activity. Nevertheless, the d-serine dehydratase activity of SHMT was physiologically important because d-serine inhibited the growth of an SHMT deletion mutant of E. coli, ∆glyA, more than that of the wild-type strain. Collectively, these results suggest that SHMT is involved not only in l- but also in d-serine metabolism through the degradation of d-serine.

Synthesis of glycine from 4-hydroxyproline in tissues of neonatal pigs.

Glycine from sow's milk only meets 20% of the requirement of suckling piglets. However, how glycine is synthesized endogenously in neonates is not known. This study determined glycine synthesis from 4-hydroxyproline (an abundant amino acid in milk and neonatal blood) in tissues of sow-reared piglets with normal birth weights. Piglets were euthanized at 0, 7, 14 and 21 days of age, and their tissues were used to determine glycine synthesis from 0 to 5 mM 4-hydroxyproline, activities and mRNA expression of key glycine-synthetic enzymes, and their cell-specific localization. Activities of 4-hydroxyproline oxidase (OH-POX), proline oxidase (POX), serine hydroxymethyltransferase (SHMT), threonine dehydrogenase (TDH), alanine:glyoxylate transaminase (AGT), and 4-hydroxy-2-oxoglutarate aldolase (HOA) occurred in the kidneys and liver from all age groups of piglets, and in the pancreas of 7- to 21-day-old piglets. Activities of OH-POX and HOA were absent from the small intestine of newborn pigs but present in the small intestine of 7- to 21-day-old piglets and in the skeletal muscle of 14- to 21-day-old piglets. Between days 0 and 21 of age, the enzymatic activities of OH-POX, AGT, and HOA decreased in the liver and kidneys but increased in the pancreas and small intestine with age. The mRNA levels of these three enzymes changed in a manner similar to their enzymatic activities. In contrast to OH-POX, AGT, and HOA, the enzymatic activities of POX, SHMT, and TDH were present in the kidneys, liver, and intestine of all age groups of piglets. Glycine was synthesized from 0.1 to 5 mM 4-hydroxyproline in the liver and kidney from 0- to 21-day-old piglets, as well as the pancreas, small intestine, and skeletal muscle from 14- to 21-day-old piglets in a concentration-dependent manner. Collectively, our findings indicate that 4-hydroxyproline is used for the synthesis of glycine in tissues of piglets to compensate for the deficiency of glycine in milk.

A gene-nutrient interaction between vitamin B6 and serine hydroxymethyltransferase (SHMT) affects genome integrity in Drosophila.

Pyridoxal 5'-phosphate (PLP), the catalytically active form of vitamin B6, participates as a cofactor to one carbon (1C) pathway that produces precursors for DNA metabolism. The concerted action of PLP-dependent serine hydroxymethyltransferase (SHMT) and thymidylate synthase (TS) leads to the biosynthesis of thymidylate (dTMP), which plays an essential function in DNA synthesis and repair. PLP deficiency causes chromosome aberrations (CABs) in Drosophila and human cells, rising the hypothesis that an altered 1C metabolism may be involved. To test this hypothesis, we used Drosophila as a model system and found, firstly, that in PLP deficient larvae SHMT activity is reduced by 40%. Second, we found that RNAi-induced SHMT depletion causes chromosome damage rescued by PLP supplementation and strongly exacerbated by PLP depletion. RNAi-induced TS depletion causes severe chromosome damage, but this is only slightly enhanced by PLP depletion. dTMP supplementation rescues CABs in both PLP-deficient and PLP-proficient SHMTRNAi . Altogether these data suggest that a reduction of SHMT activity caused by PLP deficiency contributes to chromosome damage by reducing dTMP biosynthesis. In addition, our work brings to light a gene-nutrient interaction between SHMT decreased activity and PLP deficiency impacting on genome stability that may be translated to humans.

Arabidopsis thaliana serine hydroxymethyltransferases: functions, structures, and perspectives

Serine hydroxymethyltransferase (SHM) is one of the hallmarks of one-carbon metabolism. In plants, isoforms of SHM participate in photorespiration and/or transfer the one-carbon unit from L-serine to tetrahydrofolate (THF), hence producing 5,10-CH2-THF that is needed, e.g., for biosynthesis of methionine, thymidylate, and purines. These links highlight the importance of SHM activity in DNA biogenesis, its epigenetic methylations, and in stress responses. Plant genomes encode several SHM isoforms that localize to cytosol, mitochondria, plastids, and nucleus. In this work, we present a thorough functional and structural characterization of all seven SHM isoforms from Arabidopsis thaliana (AtSHM1-7). In particular, we analyzed tissue-specific expression profiles of the AtSHM genes. We also compared catalytic properties of the active AtSHM1-4 in terms of catalytic efficiency in both directions and inhibition by the THF substrate. Despite numerous attempts to rescue the SHM activity of AtSHM5-7, we failed, which points towards different physiological functions of these isoforms. Comparative analysis of experimental and predicted three-dimensional structures of AtSHM1-7 proteins indicated differences in regions that surround the entrance to the active site cavity.

Metformin Is a Pyridoxal-5'-phosphate (PLP)-Competitive Inhibitor of SHMT2.

Simple SummaryThe mitochondrial enzyme serine hydroxymethyltransferase (SHMT2), which converts serine into glycine and generates 1C units for cell growth, is one of the most consistently overexpressed metabolic enzymes in cancer. Here, we reveal that the anti-diabetic biguanide metformin operates as a novel class of non-catalytic SHMT2 inhibitor that disrupts the pyridoxal-5′-phosphate (PLP)-dependent SHMT2 oligomerization process and ultimately SHMT2 activity. As SHMT2 inhibitors have not yet reached the clinic, these findings may aid the rational design of PLP-competitive SHMT2 inhibitors based on the biguanide skeleton of metformin.The anticancer actions of the biguanide metformin involve the functioning of the serine/glycine one-carbon metabolic network. We report that metformin directly and specifically targets the enzymatic activity of mitochondrial serine hydroxymethyltransferase (SHMT2). In vitro competitive binding assays with human recombinant SHMT1 and SHMT2 isoforms revealed that metformin preferentially inhibits SHMT2 activity by a non-catalytic mechanism. Computational docking coupled with molecular dynamics simulation predicted that metformin could occupy the cofactor pyridoxal-5′-phosphate (PLP) cavity and destabilize the formation of catalytically active SHMT2 oligomers. Differential scanning fluorimetry-based biophysical screening confirmed that metformin diminishes the capacity of PLP to promote the conversion of SHMT2 from an inactive, open state to a highly ordered, catalytically competent closed state. CRISPR/Cas9-based disruption of SHMT2, but not of SHMT1, prevented metformin from inhibiting total SHMT activity in cancer cell lines. Isotope tracing studies in SHMT1 knock-out cells confirmed that metformin decreased the SHMT2-channeled serine-to-formate flux and restricted the formate utilization in thymidylate synthesis upon overexpression of the metformin-unresponsive yeast equivalent of mitochondrial complex I (mCI). While maintaining its capacity to inhibit mitochondrial oxidative phosphorylation, metformin lost its cytotoxic and antiproliferative activity in SHMT2-null cancer cells unable to produce energy-rich NADH or FADH2 molecules from tricarboxylic acid cycle (TCA) metabolites. As currently available SHMT2 inhibitors have not yet reached the clinic, our current data establishing the structural and mechanistic bases of metformin as a small-molecule, PLP-competitive inhibitor of the SHMT2 activating oligomerization should benefit future discovery of biguanide skeleton-based novel SHMT2 inhibitors in cancer prevention and treatment.

Developmental changes in physiological amino acids and hepatic methionine remethylation enzyme activities in E10‐21 chick embryos and D1‐49 broilers

The remethylation of homocysteine to methionine is important for chick embryos to sustain the S-adenosylmethionine transmethylation reactions, which are essential for the rapid proliferation of cells. Developmental changes in hepatic 5-methyltetrahydrofolate-homocysteine methyltransferase (MFMT), betaine-homocysteine methyltransferase (BHMT) and hepatic serine hydroxymethyltransferase (SHMT) were determined in E10-21 Cobb 500 broiler chick embryos and hatched chicks from D1-49. Hepatic levels of free serine, glycine, putrescine, spermidine and spermine levels were also determined. Analyses showed hepatic MFMT-specific activity doubled from E10 to E12, with remaining embryo development experiencing small fluctuations in activity through E21. Hepatic MFMT doubled immediately after hatch, with peak activity occurring at D3. Afterwards, hepatic MFMT-specific activity steadily declined from D7-49. Hepatic BHMT activity was higher from E10 to E16 of embryogenesis, decreased rapidly at E17 and remained lower through E21 (p<.05). Hepatic BHMT-specific activity was also lower in chicks, with the exception of a peak in specific activity on D7. BHMT activity returned to lower levels by D21. Throughout embryogenesis, hepatic SHMT activity in chick embryos remained relatively constant except for a decrease at 13E, followed by an increase at 14E. Maximal activity of SHMT was found the first day post-hatch. Additionally, SHMT activity was significantly lower in growing chicks than that in embryos. Hepatic-free serine and glycine levels were negatively correlated with SHMT in hatched chicks. Hepatic polyamine, putrescine and spermidine shared a similar development pattern: peak level in the middle of incubation, low at late embryogenesis and lowest during the post-hatch period except an increase within one week after hatch. The sharp increase in hepatic concentrations of glycine, serine and putrescine, along with increased specific activities of MHMT, BHMT and SHMT from D1-7, suggests that methionine conservation (remethylation from homocysteine) and glycine/serine is critical for young chicks for organ growth, maturation, and development.

Pseudomonas syringae dual-function protein Lon switches between virulence and metabolism by acting as both DNA-binding transcriptional regulator and protease in different environments.

Lon, a member of the AAA+ protease family, plays vital roles in Type III secretion systems (T3SS), agglutination and colony shape in the model plant pathogen Pseudomonas syringae. Lon also functions as a transcriptional regulator in other bacterial species such as Escherichia coli and Brevibacillus thermoruber. To reveal the molecular mechanisms of Lon as a dual-function protein in P. syringae, we studied Lon-regulated genes by using RNA sequencing (RNA-seq), chromatin immunoprecipitation sequencing (ChIP-seq) and liquid chromatography-tandem mass spectrometry. As a transcriptional regulator, Lon directly regulated a group of genes (PSPPH_4788, gacA, fur, gntR, clpS, lon and glyA) and consequently regulated their functions, such as 1-dodecanol oxidation activity, motility, pyoverdine production, glucokinase activity, N-end rule pathway, lon expression and serine hydroxymethyltransferase activity. Mass spectrometry results revealed that the expression levels of five T3SS proteins (such as HrcV, HrpW1) were higher in the ∆lon strain than the wild-type (WT) strain in KB. In MM, 12 metabolic proteins (such as AcdS and NuoI) showed lower levels in the ∆lon strain than the WT strain. Taken together, these data demonstrate that the dual-function protein Lon sophisticatedly regulates virulence and metabolism in P. syringae.

The moonlighting RNA-binding activity of cytosolic serine hydroxymethyltransferase contributes to control compartmentalization of serine metabolism.

Enzymes of intermediary metabolism are often reported to have moonlighting functions as RNA-binding proteins and have regulatory roles beyond their primary activities. Human serine hydroxymethyltransferase (SHMT) is essential for the one-carbon metabolism, which sustains growth and proliferation in normal and tumour cells. Here, we characterize the RNA-binding function of cytosolic SHMT (SHMT1) in vitro and using cancer cell models. We show that SHMT1 controls the expression of its mitochondrial counterpart (SHMT2) by binding to the 5′untranslated region of the SHMT2 transcript (UTR2). Importantly, binding to RNA is modulated by metabolites in vitro and the formation of the SHMT1–UTR2 complex inhibits the serine cleavage activity of the SHMT1, without affecting the reverse reaction. Transfection of UTR2 in cancer cells controls SHMT1 activity and reduces cell viability. We propose a novel mechanism of SHMT regulation, which interconnects RNA and metabolites levels to control the cross-talk between cytosolic and mitochondrial compartments of serine metabolism.

Structural and functional insight into serine hydroxymethyltransferase from Helicobacter pylori.

Serine hydroxymethyltransferase (SHMT), encoded by the glyA gene, is a ubiquitous pyridoxal 5’-phosphate (PLP)-dependent enzyme that catalyzes the formation of glycine from serine. The thereby generated 5,10-methylene tetrahydrofolate (MTHF) is a major source of cellular one-carbon units and a key intermediate in thymidylate biosynthesis. While in virtually all eukaryotic and many bacterial systems thymidylate synthase ThyA, SHMT and dihydrofolate reductase (DHFR) are part of the thymidylate/folate cycle, the situation is different in organisms using flavin-dependent thymidylate synthase ThyX. Here the distinct catalytic reaction directly produces tetrahydrofolate (THF) and consequently in most ThyX-containing organisms, DHFR is absent. While the resulting influence on the folate metabolism of ThyX-containing bacteria is not fully understood, the presence of ThyX may provide growth benefits under conditions where the level of reduced folate derivatives is compromised. Interestingly, the third key enzyme implicated in generation of MTHF, serine hydroxymethyltransferase (SHMT), has a universal phylogenetic distribution, but remains understudied in ThyX-containg bacteria. To obtain functional insight into these ThyX-dependent thymidylate/folate cycles, we characterized the predicted SHMT from the ThyX-containing bacterium Helicobacter pylori. Serine hydroxymethyltransferase activity was confirmed by functional genetic complementation of a glyA-inactivated E. coli strain. A H. pylori ΔglyA strain was obtained, but exhibited markedly slowed growth and had lost the virulence factor CagA. Biochemical and spectroscopic evidence indicated formation of a characteristic enzyme-PLP-glycine-folate complex and revealed unexpectedly weak binding affinity of PLP. The three-dimensional structure of the H. pylori SHMT apoprotein was determined at 2.8Ǻ resolution, suggesting a structural basis for the low affinity of the enzyme for its cofactor. Stabilization of the proposed inactive configuration using small molecules has potential to provide a specific way for inhibiting HpSHMT.

The catalytic activity of serine hydroxymethyltransferase is essential for denovo nuclear dTMP synthesis in lung cancer cells.

Structural data are available in the PDB under the accession number 6FL5.

Mitochondrial translation requires folate-dependent tRNA methylation

Folates enable the activation and transfer of one-carbon units for biosynthesis of purines, thymidine and methionine1–3. Antifolates are important immunosuppressive4 and anticancer agents5. In proliferating lymphocytes6 and human cancers7,8, folate enzymes localizing to the mitochondria are particularly strongly upregulated. This in part reflects the need for mitochondria to generate one-carbon units and export them to the cytosol for anabolic metabolism2,9. The full range of uses of folate-bound one-carbon units in the mitochondrial compartment itself, however, has not been thoroughly explored. Here we show that loss of catalytic activity of the mitochondrial folate enzyme serine hydroxymethyltransferase 2 (SHMT2), but not other folate enzymes, leads to defective oxidative phosphorylation due to impaired mitochondrial translation. We find that SHMT2, presumably by generating mitochondrial 5,10-methylenetetrahydrofolate, provides methyl donors for producing the taurinomethyluridine base at the wobble position of select mitochondrial tRNAs. Mitochondrial ribosome profiling reveals that SHMT2 knockout cells, due to lack of this modified base, suffer from defective translation with preferential mitochondrial ribosome stalling at certain lysine (AAG) and leucine (UUG) codons. This results in impaired respiratory chain enzyme expression. Stalling at these specific codons also occurs in certain mitochondrial inborn errors of metabolism. Disrupting whole-cell folate metabolism, by folate deficiency or antifolate therapy, also impairs the respiratory chain. In summary, mammalian mitochondria use folate-bound one-carbon units to methylate tRNA, and this modification is required for respiratory chain translation and thus oxidative phosphorylation.

A pyrazolopyran derivative preferentially inhibits the activity of human cytosolic serine hydroxymethyltransferase and induces cell death in lung cancer cells.

Serine hydroxymethyltransferase (SHMT) is a central enzyme in the metabolic reprogramming of cancer cells, providing activated one-carbon units in the serine-glycine one-carbon metabolism. Previous studies demonstrated that the cytoplasmic isoform of SHMT (SHMT1) plays a relevant role in lung cancer. SHMT1 is overexpressed in lung cancer patients and NSCLC cell lines. Moreover, SHMT1 is required to maintain DNA integrity. Depletion in lung cancer cell lines causes cell cycle arrest and uracil accumulation and ultimately leads to apoptosis. We found that a pyrazolopyran compound, namely 2.12, preferentially inhibits SHMT1 compared to the mitochondrial counterpart SHMT2. Computational and crystallographic approaches suggest binding at the active site of SHMT1 and a competitive inhibition mechanism. A radio isotopic activity assay shows that inhibition of SHMT by 2.12 also occurs in living cells. Moreover, administration of 2.12 in A549 and H1299 lung cancer cell lines causes apoptosis at LD50 34 μM and rescue experiments underlined selectivity towards SHMT1. These data not only further highlight the relevance of the cytoplasmic isoform SHMT1 in lung cancer but, more importantly, demonstrate that, at least in vitro, it is possible to find selective inhibitors against one specific isoform of SHMT, a key target in metabolic reprogramming of many cancer types.

Characterization of a serine hydroxymethyltransferase for l-serine enzymatic production from Pseudomonas plecoglossicida

Pseudomonas plecoglossicida, a bacterium strain that exhibits high Serine hydroxymethyltransferase (SHMT) activity, was isolated from the seawater. A full-length glyA encoding SHMT was obtained by a modified thermal asymmetric interlaced-PCR (TRIL-PCR), which consisted of 1,254 bp, encoded a 417 amino acid polypeptide, and shared the highest identity (75 %) with a glyA gene from Acinetobacter radioresistens CMC-1. Recombinant glyA gene was expressed in Escherichia coli BL21 (DE3) and purified by electrophoretic homogeneity. The enzyme showed the optimal activity at pH 8.0 and 40 °C, and remained stable in high alkali conditions. Using SHMT to produce L-serine by catalyzing the reaction of glycine and tetrahydrofolate is one of the most promising routes to synthesize L-serine, achieving 33.4 mM L-serine at the 12th h of the enzymatic reaction with the substrates of glycine (133 mM) and formaldehyde (13.3 mM). The properties make the SHMT a candidate for further enzymatic studies and industrial applications.

A serine hydroxymethyltransferase from marine bacterium Shewanella algae: Isolation, purification, characterization and l-serine production

Currently, l-serine is mainly produced by enzymatic conversion, in which serine hydroxymethyltransferase (SHMT) is the key enzyme, suggesting the importance of searching for a SHMT with high activity. Shewanella algae, a methanol-utilizing marine bacterium showing high SHMT activity, was selected based on screening bacterial strains and comparison of the activities of SHMTs. A glyA was isolated from the S. algae through thermal asymmetric interlaced PCR (TAIL-PCR) and it encoded a 417 amino acid polypeptide. The SaSHMT, encoded by the glyA, showed the optimal activity at 50°C and pH 7.0, and retained over 45% of its maximal activity after incubation at 40°C for 3h. The enzyme showed better stability under alkaline environment (pH 6.5–9.0) than Hyphomicrobium methylovorum GM2's SHMT (pH 6.0–7.5). The SaSHMT can produce 77.76mM of l-serine by enzymatic conversion, with the molecular conversion rate in catalyzing glycine to l-serine being 1.41-fold higher than that of Escherichia coli. Therefore, the SaSHMT has the potential for industrial applications due to its tolerance of alkaline environment and a relatively high enzymatic conversion rate.

Glycine consumption and mitochondrial serine hydroxymethyltransferase in cancer cells: The heme connection

It was recently discovered that glycine consumption is strongly related to the rate of proliferation across cancer cells. This is very intriguing and raises the question of what is the actual role of this amino acid in cancer metabolism. Cancer cells are greedy for glycine. In particular, the mitochondrial production of glycine seems to be utterly important. Overexpression of mitochondrial serine hydroxymethyltransferase, the enzyme converting l-serine to glycine, assures an adequate supply of glycine to rapidly proliferating cancer cells. In fact, silencing of mitochondrial serine hydroxymethyltransferase was shown to halt cancer cell proliferation. Direct incorporation of glycine carbon atoms into the purine ring has been proposed to be one main reason for the importance of glycine in cancer cell metabolism.We believe that, as far as the importance of glycine in cancer is concerned, a central role of this amino acid, namely its participation to heme biosynthesis, has been neglected. In mitochondria, glycine condenses with succinyl-CoA to form 5-aminolevulinate, the universal precursor of the different forms of heme contained in cytochromes and oxidative phosphorylation complexes.Our hypothesis is that mitochondrial serine hydroxymethyltransferase is fundamental to sustain cancer metabolism since production of glycine fuels heme biosynthesis and therefore oxidative phosphorylation. Respiration of cancer cells may then ultimately rely on endogenous glycine synthesis by mitochondrial serine hydroxymethyltransferase. The link between mitochondrial serine hydroxymethyltransferase activity and heme biosynthesis represents an important and still unexplored aspect of the whole picture of cancer cell metabolism.Our hypothesis might be tested using a combination of metabolic tracing and gene silencing on different cancer cell lines. The experiments should be devised so as to assess the importance of mitochondrial serine hydroxymethyltransferase and the glycine deriving from its reaction as a precursor of heme.If the observed increase of glycine consumption in rapidly proliferating cancer cells has its basis in the need for heme biosynthesis, then mitochondrial serine hydroxymethyltransferase should be considered as a key target for the development of new chemotherapic agents.

Is the recovery of (photo) respiratory CO2 and intermediates minimal?

Is the recovery of (photo) respiratory <scp>CO</scp>₂ and intermediates minimal?

Plasma Pyridoxal-5-Phosphate Is Inversely Associated with Systemic Markers of Inflammation in a Population of U.S. Adults1–3

Low vitamin B-6 status, based on plasma concentrations of pyridoxal-5-phosphate (PLP), has been identified in inflammatory diseases, including cardiovascular disease, rheumatoid arthritis, inflammatory bowel disease, and diabetes. Our objective was to examine the association between plasma PLP and multiple markers of inflammation in a community-based cohort [n = 2229 participants (55% women, mean age 61 #x00B1; 9 y)]. We created an overall inflammation score (IS) as the sum of standardized values of 13 individual inflammatory markers. Multivariable-adjusted regression analysis was used to assess the associations between the IS and plasma PLP. Geometric mean plasma PLP concentrations were lower in the highest tertile category of IS relative to the lowest (61 vs. 80 nmol/L; P-trend < 0.0001). Similarly, the prevalence of PLP insufficiency was significantly higher for participants in the highest compared with the lowest tertiles for IS categories. These relationships persisted after accounting for vitamin B-6 intake. Also, there were significant inverse relationships between plasma PLP and 4 IS based on functionally related markers, including acute phase reactants, cytokines, adhesion molecules, and oxidative stress. In addition, secondary analyses revealed that many of the individual inflammatory markers were inversely associated with plasma PLP after adjusting for plasma C-reactive protein concentration. This study, in combination with past findings, further supports our hypothesis that inflammation is associated with a functional deficiency of vitamin B-6. We discuss 2 possible roles for PLP in the inflammatory process, including tryptophan metabolism and serine hydroxymethyltransferase activity.

Plasmodium serine hydroxymethyltransferase as a potential anti-malarial target: inhibition studies using improved methods for enzyme production and assay

BackgroundThere is an urgent need for the discovery of new anti-malarial drugs. Thus, it is essential to explore different potential new targets that are unique to the parasite or that are required for its viability in order to develop new interventions for treating the disease. Plasmodium serine hydroxymethyltransferase (SHMT), an enzyme in the dTMP synthesis cycle, is a potential target for such new drugs, but convenient methods for producing and assaying the enzyme are still lacking, hampering the ability to screen inhibitors.MethodsProduction of recombinant Plasmodium falciparum SHMT (PfSHMT) and Plasmodium vivax SHMT (PvSHMT), using auto-induction media, were compared to those using the conventional Luria Bertani medium with isopropyl thio-β-D-galactoside (LB-IPTG) induction media. Plasmodium SHMT activity, kinetic parameters, and response to inhibitors were measured spectrophotometrically by coupling the reaction to that of 5,10-methylenetetrahydrofolate dehydrogenase (MTHFD). The identity of the intermediate formed upon inactivation of Plasmodium SHMTs by thiosemicarbazide was investigated by spectrophotometry, high performance liquid chromatography (HPLC), and liquid chromatography-mass spectrometry (LC-MS). The active site environment of Plasmodium SHMT was probed based on changes in the fluorescence emission spectrum upon addition of amino acids and folate.ResultsAuto-induction media resulted in a two to three-fold higher yield of Pf- and PvSHMT (7.38 and 29.29 mg/L) compared to that produced in cells induced in LB-IPTG media. A convenient spectrophotometric activity assay coupling Plasmodium SHMT and MTHFD gave similar kinetic parameters to those previously obtained from the anaerobic assay coupling SHMT and 5,10-methylenetetrahydrofolate reductase (MTHFR); thus demonstrating the validity of the new assay procedure. The improved method was adopted to screen for Plasmodium SHMT inhibitors, of which some were originally designed as inhibitors of malarial dihydrofolate reductase. Plasmodium SHMT was slowly inactivated by thiosemicarbazide and formed a covalent intermediate, PLP-thiosemicarbazone.ConclusionsAuto-induction media offers a cost-effective method for the production of Plasmodium SHMTs and should be applicable for other Plasmodium enzymes. The SHMT-MTHFD coupled assay is equivalent to the SHMT-MTHFR coupled assay, but is more convenient for inhibitor screening and other studies of the enzyme. In addition to inhibitors of malarial SHMT, the development of species-specific, anti-SHMT inhibitors is plausible due to the presence of differential active sites on the Plasmodium enzymes.