Cytosolic Serine Hydroxymethyltransferase Research Articles

Contribution of mitochondrial serine and glycine‐derived one‐carbon units to deoxynucleoside synthesis in humans

BACKGROUND Serine and glycine are major one-carbon donors. In eukaryotic cells one-carbon metabolism is compartmentalized; the glycine cleavage enzymes are mitochondrial, while the cytosolic and mitochondrial forms of serine hydroxymethyltransferase (SHMT) are independently transcribed. DESIGN One-carbon donors ([3-13C]serine, [2, 3, 3-d3]serine, or [2-13C]glycine) were administered in separate infusions to healthy males (n = 5). Monocyte DNA was subsequently isolated for the determination of deoxynucleotide enrichment by LC-MS/MS. RESULTS Total thymidine and deoxyadenosine enrichment was similar for [3-13C]serine and [2, 3, 3-d3]serine [Table]. M+2 thymidine enrichment (i.e., that deriving from cytosolic SHMT) constituted 23% of [2, 3, 3-d3]serine derived enrichment. Thymidine enrichment from [2-13C]glycine was lower than from either labeled serine while deoxyadenosine enrichment from [2-13C]glycine was overestimated, as [2-13C]glycine was also directly incorporated into purines. Percentage molar enrichment (mean ± SD) of thymidine (dT) and deoxyadenosine (dA). Values in brackets are molar enrichments expressed as a percentage of total one-carbon enrichment: {enrichment / (enrichment from [2, 3, 3-d3]serine + enrichment from [2-13C]glycine)} CONCLUSION The majority (~84%) of one-carbon units used for thymidine synthesis are mitochondrial derived. Supported by NIH DK56274 and GCRC grant M01-RR00082

Cloning, expression, purification, and characterization of zebrafish cytosolic serine hydroxymethyltransferase

A cDNA which encodes for zebrafish serine hydroxymethyltransferase (SHMT) has been cloned into a pET43.1a vector as a NdeI– EcoRI insert and transformed into HMS174(DE3) cells. After induction with isopropyl thiogalactoside, the enzyme was purified with a three-step purification protocol and about 15 mg of pure enzyme was obtained per liter of culture. Spectral and structural characteristics of the recombinant zebrafish SHMT are similar to the rabbit and human cytosolic SHMT. Kinetic constants for the natural substrates l-serine and tetrahydrofolate are also comparable to the values obtained previously for the rabbit and human cytosolic enzyme.

Properties of human and rabbit cytosolic serine hydroxymethyltransferase are changed by single nucleotide polymorphic mutations

Serine hydroxymethyltransferase (SHMT) is a key enzyme in the formation and regulation of the folate one-carbon pool. Recent studies on human subjects have shown the existence of two single nucleotide polymorphisms that may be associated with several disease states. One of these mutations results in Ser394 being converted to an Asn (S394N) and the other in the change of Leu474 to a Phe (L474F). These mutations were introduced into the cDNA for both human and rabbit cytosolic SHMT and the mutant enzymes expressed and purified from an Escherichia coli expression system. The mutant enzymes show normal values for k cat and K m for serine. However, the S394N mutant enzyme has increased dissociation constant values for both glycine and tetrahydrofolate (tetrahydropteroylglutamate) and its pentaglutamate form compared to wild-type enzyme. The L474F mutant shows lowered affinity (increased dissociation constant) for only the pentaglutamate form of the folate ligand. Both mutations result in decreased rates of pyridoxal phosphate addition to the mutant apo enzymes to form the active holo enzymes. Neither mutation significantly affects the stability of SHMT or the rate at which it converts 5,10-methenyl tetrahydropteroyl pentaglutamate to 5-formyl tetrahydropteroyl pentaglutamate. Analysis of the structures of rabbit and human SHMT show how mutations at these two sites can result in the observed functional differences.

Polymorphisms and haplotypes of serine hydroxymethyltransferase and risk of squamous cell carcinoma of the head and neck: a case–control analysis

Low dietary intake of fruits and vegetables, particularly folate deficiency, has been associated with the risk of squamous cell carcinoma of the head and neck (SCCHN). We hypothesized that polymorphisms of the cytosolic serine hydroxymethyltransferase (SHMT1) gene involved in folate-dependent, one-carbon metabolism are associated with SCCHN risk. In a hospital-based, case-control study of 721 non-Hispanic white SCCHN patients and 1,234 control subjects, frequency-matched by age and sex, three known SHMT1 polymorphisms (34761C>T, 34840C>G and 34859C>T) were genotyped. It was found that none of these three polymorphisms alone had a significant main effect on the risk of SCCHN. However, when the three polymorphisms were evaluated together by the number of the variant (risk) haplotype alleles (i.e. 34761 T, 34840G or 34859 T), the risk of SCCHN was significantly increased in a dose-response manner as the number of variant haplotype alleles increased compared to those with zero variant alleles [adjusted odd ratio (OR)=1.39, 95% confidence interval (CI)=1.14-1.70 for 1-3 variant alleles and OR=1.46, 95% CI=1.09-1.97 for 4-6 variant alleles; Ptrend=0.001]. In stratification analysis, this significant association was confined to younger men (<or=64 years), ever smokers, ever drinkers and patients whose primary tumor site was in the pharynx or larynx. In conclusion, these three SHMT1 polymorphisms may play a joint role in the etiology of SCCHN in a non-Hispanic white population.

Increased expression of cSHMT, Tbx3 and utrophin in plasma of ovarian and breast cancer patients

Plasma samples of ovarian and breast cancer patients were used to search for markers of cancer using 2-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry. Truncated forms of cytosolic serine hydroxymethyl transferase (cSHMT), T-box transcription factor 3 (Tbx3) and utrophin were aberrantly expressed in samples from cancer patients as compared to samples from noncancerous cases. Aberrant expression of proteins was validated by immunoblotting of plasma samples with specific antibodies to cSHMT, Tbx3 and utrophin. A cohort of 79 breast and 39 ovarian cancer patients and 31 individuals with noncancerous conditions was studied. We observed increased expression of truncated cSHMT, Tbx3 and utrophin in plasma samples obtained from patients at early stages of disease. Our data suggest that cSHMT, Tbx3 and utrophin can be used as components of multiparameter monitoring of ovarian and breast cancer (supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat/index.html).

Activities of Hepatic Cytosolic and Mitochondrial Forms of Serine Hydroxymethyltransferase and Hepatic Glycine Concentration Are Affected by Vitamin B-6 Intake in Rats

Serine hydroxymethyltransferase (SHMT) is a pyridoxal phosphate (PLP)–dependent enzyme that exists as cytosolic and mitochondrial isozymes that catalyze the reversible interconversion of serine and tetrahydrofolate (THF) to glycine and 5,10-methyleneTHF. SHMT is a major source of one-carbon units for cellular metabolism, but its sensitivity to various degrees of altered vitamin B-6 nutritional status has not been determined. In this study, cytosolic and mitochondrial SHMT activities were measured in liver from rats fed dietary pyridoxine (PN) ranging from adequate to deficient levels (2, 1, 0.5, 0.1, and 0 mg PN/kg diet; n = 10 per group). Both mitochondrial and cytosolic SHMT activities increased (P < 0.001) with increasing dietary PN over this range, and activities were a linear function of liver PLP concentration. Mitochondrial SHMT comprised ∼70% of total activity. Assays conducted with and without in vitro addition of PLP indicated that total SHMT (apo- and holoenzyme forms) varied with dietary PN for each isoform, but that the proportion of each present as the apoenzyme was not affected by PN intake. This aspect of SHMT nutritional regulation differs from that of many other PLP-dependent enzymes. Hepatic glycine concentration was inversely related to vitamin B-6 intake (P < 0.05), which suggests a functional effect of altered SHMT activity. Overall these results demonstrate the potential for disruption of SHMT-mediated one-carbon metabolism by inadequate vitamin B-6 intake.

A three-dimensional structure of Plasmodium falciparum serine hydroxymethyltransferase in complex with glycine and 5-formyl-tetrahydrofolate. Homology modeling and molecular dynamics

Cytosolic Plasmodium falciparum serine hydroxymethyltransferase (pfSHMT) is a potential target for antimalarial chemotherapy. Contrasting with the other enzymes involved in the parasite folate cycle, little information is available about this enzyme, and its crystallographic structure is unknown yet. In this paper, we propose a theoretical low-resolution 3D model for pfSHMT in complex with glycine and 5-formyl tetrahydrofolate (5-FTHF) based on homology modeling by multiple alignment followed by intensive optimization, validation and dynamics simulations in water. Comparison between the active sites of our model and that of crystallographic Human SHMT (hSHMT) revealed key differences that could be useful for the design of new selective inhibitors of pfSHMT.

Alanine : glyoxylate aminotransferase of Saccharomyces cerevisiae-encoding gene AGX1 and metabolic significance.

Alanine : glyoxylate aminotransferase is one of three different enzymes used for glycine synthesis in Saccharomyces cerevisiae. The open reading frame YFL030w (named AGX1 in the following), encoding this enzyme, was identified by comparing enzyme specific activities in knockout strains. While 100% activity was detectable in the parental strain, 2% was found in a YFL030w::kanMX4 strain. The ORF found at that locus was suspected to encode alanine : glyoxylate aminotransferase because its predicted amino acid sequence showed 23% identity to the human homologue. Since the YFL030w::kanMX4 strain showed no glycine auxtrophic phenotype, AGX1 was replaced by KanMX4 in a Delta GLY1 Delta SHM1 Delta SHM2 background. These background mutations, which cause inactivation of threonine aldolase, mitochondrial and cytosolic serine hydroxymethyltransferase, respectively, lead to a conditional glycine auxotrophy. This means that growth is not possible on glucose but on ethanol as the sole carbon source. Additional disruption of AGX1 revealed a complete glycine auxotrophy. Complementation was observed by transformation with a plasmid-encoded AGX1.

Genetic Interaction between a Chaperone of Small Nucleolar Ribonucleoprotein Particles and Cytosolic Serine Hydroxymethyltransferase

Srp40p is a nonessential yeast nucleolar protein proposed to function as a chaperone for over 100 small nucleolar ribonucleoprotein particles that are required for rRNA maturation. To verify and expand on its function, genetic screens were performed for the identification of genes that were lethal when mutated in a SRP40 null background (srp40Delta). Unexpectedly, mutation of both cytosolic serine hydroxymethyltransferase (SHM2) and one-carbon tetrahydrofolate synthase (ADE3) was required to achieve synthetic lethality with srp40Delta. Shm2p and Ade3p are cytoplasmic enzymes producing 5,10-methylene tetrahydrofolate in convergent pathways as the primary source for cellular one-carbon groups. Nonetheless, point mutants of Shm2p that were catalytically inactive (i.e. failed to rescue the methionine auxotrophy of a shm2Delta ade3 strain) complemented the synthetic lethal phenotype, thus revealing a novel metabolism-independent function of Shm2p. The same Shm2p mutants exacerbated a giant cell phenotype observed in the shm2Delta ade3 strain suggesting a catalysis-independent role for Shm2p in cell size control, possibly through regulation of ribosome biogenesis via SRP40. Additionally, we show that the Sm-like protein Lsm5p, which as part of Lsm complexes participates in cytosolic and nuclear RNA processing and degradation pathways, is a multicopy suppressor of the synthetic lethality and of the specific depletion of box H/ACA small nucleolar RNAs from the srp40Delta shm2 ade3 strain. Finally, rat Nopp140 restored growth and stability of box H/ACA snoRNAs after genetic depletion of SRP40 in the synthetic lethal strain indicating that it is indeed the functional homolog of yeast Srp40p.

Identification of amino acid residues, essential for maintaining the tetrameric structure of sheep liver cytosolic serine hydroxymethyltransferase, by targeted mutagenesis.

Serine hydroxymethyltransferase (SHMT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, catalyses the transfer of the hydroxymethyl group from serine to tetrahydrofolate to yield glycine and N (5), N (10)-methylenetetrahydrofolate. An analysis of the known SHMT sequences indicated that several amino acid residues were conserved. In this paper, we report the identification of the amino acid residues essential for maintaining the oligomeric structure of sheep liver cytosolic recombinant SHMT (scSHMT) through intra- and inter-subunit interactions and by stabilizing the binding of PLP at the active site. The mutation of Lys-71, Arg-80 and Asp-89, the residues involved in intra-subunit ionic interactions, disturbed the oligomeric structure and caused a loss of catalytic activity. Mutation of Trp-110 to Phe was without effect, while its mutation to Ala resulted in the enzyme being present in the insoluble fraction. These results suggested that Trp-110 located in a cluster of hydrophobic residues was essential for proper folding of the enzyme. Arg-98 and His-304, residues involved in the inter-subunit interactions, were essential for maintaining the tetrameric structure. Mutation of Tyr-72, Asp-227 and His-356 at the active site which interact with PLP resulted in the loss of PLP, and hence loss of tetrameric structure. Mutation of Cys-203, located away from the active site, weakened PLP binding indirectly. The results demonstrate that in addition to residues involved in inter-subunit interactions, those involved in PLP binding and intra-subunit interactions also affect the oligomeric structure of scSHMT.

Location of the Pteroylpolyglutamate-binding Site on Rabbit Cytosolic Serine Hydroxymethyltransferase

Serine hydroxymethyltransferase (SHMT; EC 2.1.2.1) catalyzes the reversible interconversion of serine and glycine with transfer of the serine side chain one-carbon group to tetrahydropteroylglutamate (H(4)PteGlu), and also the conversion of 5,10-methenyl-H(4)PteGlu to 5-formyl-H(4)PteGlu. In the cell, H(4)PteGlu carries a poly-gamma-glutamyl tail of at least 3 glutamyl residues that is required for physiological activity. This study combines solution binding and mutagenesis studies with crystallographic structure determination to identify the extended binding site for tetrahydropteroylpolyglutamate on rabbit cytosolic SHMT. Equilibrium binding and kinetic measurements of H(4)PteGlu(3) and H(4)PteGlu(5) with wild-type and Lys --> Gln or Glu site mutant homotetrameric rabbit cytosolic SHMTs identified lysine residues that contribute to the binding of the polyglutamate tail. The crystal structure of the enzyme in complex with 5-formyl-H(4)PteGlu(3) confirms the solution data and indicates that the conformation of the pteridine ring and its interactions with the enzyme differ slightly from those observed in complexes of the monoglutamate cofactor. The polyglutamate chain, which does not contribute to catalysis, exists in multiple conformations in each of the two occupied binding sites and appears to be bound by the electrostatic field created by the cationic residues, with only limited interactions with specific individual residues.

Overexpression and characterization of dimeric and tetrameric forms of recombinant serine hydroxymethyltransferase from Bacillus stearothermophilus.

Serine hydroxymethyltransferase (SHMT), a pyridoxal-5' -phosphate (PLP) dependent enzyme catalyzes the interconversion of L-Ser and Gly using tetrahydrofolate as a substrate. The gene encoding for SHMT was amplified by PCR from genomic DNA of Bacillus stearothermophilus and the PCR product was cloned and overexpressed in Escherichia coli. The purified recombinant enzyme was isolated as a mixture of dimer (90%) and tetramer (10%). This is the first report demonstrating the existence of SHMT as a dimer and tetramer in the same organism. The specific activities at 37 C of the dimeric and tetrameric forms were 6 7 U/mg and 4 1 U/mg, respectively. The purified dimer was extremely thermostable with a T(m) of 85 degrees C in the presence of PLP and L-Ser. The temperature optimum of the dimer was 80 degrees C with a specific activity of 32 4 U/mg at this temperature. The enzyme catalyzed tetrahydrofolate-independent reactions at a slower rate compared to the tetrahydrofolate-dependent retro-aldol cleavage of L-Ser. The interaction with substrates and their analogues indicated that the orientation of PLP ring of B. stearothermophilus SHMT was probably different from sheep liver cytosolic recombinant SHMT (scSHMT).

Polymorphisms in the thymidylate synthase and serine hydroxymethyltransferase genes and risk of adult acute lymphocytic leukemia

We previously reported that 2 polymorphisms in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene at positions C677T and A1298C were associated with lower risk of adult acute lymphocytic leukemia (ALL). In the present study, we have examined whether polymorphisms in other folate-metabolizing genes play a role in ALL susceptibility. Polymorphisms in methionine synthase (MS A2756G), cytosolic serine hydroxymethyltransferase (SHMT1 C1420T), and a double (2R2R) or triple (3R3R) 28-bp tandem repeat in the promoter region of thymidylate synthase (TS) were studied and found to modulate ALL risk. In a univariate analysis, SHMT1 1420CT individuals exhibited a 2.1-fold decrease in ALL risk (odds ratio [OR] = 0.48; 95% confidence interval [CI], 0.25-0.91), whereas the 1420TT genotype conferred a 3.3-fold reduction in risk (OR = 0.31; 95% CI, 0.10-0.90). Similarly, TS 2R3R individuals exhibited a 2.8-fold reduction in ALL risk (OR = 0.36; 95% CI: 0.16-0.83), while the TS 3R3R genotype conferred an even greater level of protection (OR = 0.25; 95% CI, 0.08-0.78). However, no significant associations were evident for the MS 2756AG polymorphism (OR = 0.79; 95% CI, 0.38-1.7). In addition, potential interactions between the SHMT1 and TS or MS genes were observed. TS 3R3R individuals who were SHMT1 1420CT/TT had a 13.9-fold decreased ALL risk (OR = 0.072; 95% CI, 0.0067-0.77). Further, MS 2756AG individuals who were SHMT1 1420CT/TT had a 5.6-fold reduction in ALL risk (OR = 0.18; 95% CI, 0.05-0.63). This study suggests an important role for uracil misincorporation and resultant chromosomal damage in the pathogenesis of ALL, and that genetic interactions involving low penetrance polymorphisms in folate-metabolizing genes may increase ALL risk.

The Role of Serine Hydroxymethyltransferase Isozymes in One-Carbon Metabolism in MCF-7 Cells as Determined by 13C NMR

The role of cytosolic and mitochondrial serine hydroxymethyltransferase in supplying one-carbon groups for purine and thymidylate biosynthesis in MCF-7 cells was investigated by observing folate-mediated one-carbon metabolism of l-[3-13C]serine, [2-13C]glycine, and [13C]formate. 13C NMR was used to follow the incorporation of label into carbons 2 and 8 of purines and the methyl group attached to carbon 5 of thymidylate. The percentage enrichment of the 13C label in purines was determined from the splitting patterns of the 1H NMR spectra of C2 and C8 of adenine and C8 of guanine. The results show that formate is the major precursor in the cytosol of the one-carbon group in 10-formyltetrahydrofolate, which is used in purine biosynthesis, and the one-carbon group in 5,10-methylenetetrahydrofolate, which is used in thymidylate biosynthesis. Formate is formed in the mitochondria from carbon 3 of serine. The cleavage of serine to glycine and 5,10-methylenetetrahydrofolate by cytosolic serine hydroxymethyltransferase does not appear to be a major source of one-carbon groups for either purine or thymidylate biosynthesis. Carbon 3 of serine accounts for about 95% of the one-carbon pool, suggesting that other sources of one-carbon groups represent only minor pathways. [2-13C]Glycine is not a donor of one-carbons groups, confirming that MCF-7 cells lack a functional glycine cleavage system.

Ontogeny of hepatic enzymes involved in serine- and folate-dependent one-carbon metabolism in rabbits.

Serine occupies a central position in folate-dependent, one-carbon metabolism through 5,10-methylenetetrahydrofolate (MTHF) and 5-formyltetrahydrofolate (FTHF). We characterized the ontogeny of the specific activity of key enzymes involved in serine, 5,10-MTHF, and 5-FTHF metabolism: methenyltetrahydrofolate synthetase (MTHFS), MTHF reductase (MTHFR), the glycine cleavage system (GCS), methionine synthase (MS), and serine hydroxymethyltransferase (SHMT) in rabbit liver, placenta, brain, and kidney. In liver, MTHFS activity is low in the fetus (0.36 +/- 0.07 nmol. min(-1). mg protein(-1)), peaks at 3 wk (1.48 +/- 0.50 nmol. min(-1). mg protein(-1)), and then decreases to adult levels (1.13 +/- 0.32 nmol. min(-1). mg protein(-1)). MTHFR activity is highest early in gestation (24.9 +/- 2.4 nmol. h(-1). mg protein(-1)) and declines rapidly by birth (4.7 +/- 1.3 nmol. h(-1). mg protein(-1)). MS is highest during fetal life and declines after birth. Cytosolic SHMT activity does not vary during development, but mitochondrial SHMT peaks at 23 days. GCS activity is high in the fetus and the neonate, declining after weaning. In placenta and brain, all activities are low throughout gestation. Cytosolic and mitochondrial SHMT activities are low in kidney and rise after weaning, whereas MTHFS is low throughout development. These data suggest that the liver is the primary site of activity for these enzymes. Throughout development, there are multiple potential sources for production of 5,10-MTHF, but early in gestation high MTHFR activity and low MTHFS activity could reduce 5,10-MTHF availability.

The role of Glu74 and Tyr82 in the reaction catalyzed by sheep liver cytosolic serine hydroxymethyltransferase.

The three-dimensional structures of human and rabbit liver cytosolic recombinant serine hydroxymethyltransferases (hcSHMT and rcSHMT) revealed that E75 and Y83 (numbering according to hcSHMT) are probable candidates for proton abstraction and Calpha-Cbeta bond cleavage in the reaction catalyzed by serine hydroxymethyltransferase. Both these residues are completely conserved in all serine hydroxymethyltransferases sequenced to date. In an attempt to decipher the role of these residues in sheep liver cytosolic recombinant serine hydroxymethyltransferase (scSHMT), E74 (corresponding residue is E75 in hcSHMT) was mutated to Q and K, and Y82 (corresponding residue is Y83 in hcSHMT) was mutated to F. The specific activities using serine as the substrate for the E74Q and E74K mutant enzymes were drastically reduced. These mutant enzymes catalyzed the transamination of D-alanine and 5,6,7, 8-tetrahydrofolate independent retroaldol cleavage of Lallo threonine at rates comparable with wild-type enzyme, suggesting that E74 was not involved directly in the proton abstraction step of catalysis, as predicted earlier from crystal structures of hcSHMT and rcSHMT. There was no change in the apparent Tm value of E74Q upon the addition of L-serine, whereas the apparent Tm value of scSHMT was enhanced by 10 degrees C. Differential scanning calorimetric data and proteolytic digestion patterns in the presence of L-serine showed that E74Q was different to scSHMT. These results indicated that E74 might be required for the conformational change involved in reaction specificity. It was predicted from the crystal structures of hcSHMT and rcSHMT that Y82 was involved in hemiacetal formation following Calpha-Cbeta bond cleavage of L-serine and mutation of this residue to F could lead to a rapid release of HCHO. However, the Y82F mutant had only 5% of the activity and failed to form a quinonoid intermediate, suggesting that this residue is not involved in the formation of the hemiacetal intermediate, but might be involved indirectly in the abstraction of the proton and in stabilizing the quinonoid intermediate.

Role of Pro-297 in the catalytic mechanism of sheep liver serine hydroxymethyltransferase

Serine hydroxymethyltransferase belongs to the α class of pyridoxal-5´-phosphate enzymes along with aspartate aminotransferase. Recent reports on the three-dimensional structure of human liver cytosolic serine hydroxymethyltransferase had suggested a high degree of similarity between the active-site geometries of the two enzymes. A comparison of the sequences of serine hydroxymethyltransferases revealed the presence of several highly conserved residues, including Pro-297. This residue is equivalent to residue Arg-292 of aspartate aminotransferase, which binds the γ-carboxy group of aspartate. In an attempt to change the reaction specificity of the hydroxymethyltransferase to that of an aminotransferase and to assign a possible reason for the conserved nature of Pro-297, it was mutated to Arg. The mutation decreased the hydroxymethyltransferase activity significantly (by 85–90%) and abolished the ability to catalyse alternative reactions, without alteration in the oligomeric structure, pyridoxal 5´-phosphate content or substrate binding. However, the concentration of the quinonoid intermediate and the extent of proton exchange was decreased considerably (by approx. 85%) corresponding to the decrease in catalytic activity. Interestingly, mutant Pro-297 Arg was unable to perform the transamination reaction with l-aspartate. All these results suggest that although Pro-297 is indirectly involved in catalysis, it might not have any role in imparting substrate specificity, unlike the similarly positioned Arg-292 in aspartate aminotransferase.

Role of Pro-297 in the catalytic mechanism of sheep liver serine hydroxymethyltransferase

Serine hydroxymethyltransferase belongs to the alpha class of pyridoxal-5'-phosphate enzymes along with aspartate aminotransferase. Recent reports on the three-dimensional structure of human liver cytosolic serine hydroxymethyltransferase had suggested a high degree of similarity between the active-site geometries of the two enzymes. A comparison of the sequences of serine hydroxymethyltransferases revealed the presence of several highly conserved residues, including Pro-297. This residue is equivalent to residue Arg-292 of aspartate aminotransferase, which binds the gamma-carboxy group of aspartate. In an attempt to change the reaction specificity of the hydroxymethyltransferase to that of an aminotransferase and to assign a possible reason for the conserved nature of Pro-297, it was mutated to Arg. The mutation decreased the hydroxymethyltransferase activity significantly (by 85-90%) and abolished the ability to catalyse alternative reactions, without alteration in the oligomeric structure, pyridoxal 5'-phosphate content or substrate binding. However, the concentration of the quinonoid intermediate and the extent of proton exchange was decreased considerably (by approx. 85%) corresponding to the decrease in catalytic activity. Interestingly, mutant Pro-297 Arg was unable to perform the transamination reaction with L-aspartate. All these results suggest that although Pro-297 is indirectly involved in catalysis, it might not have any role in imparting substrate specificity, unlike the similarly positioned Arg-292 in aspartate aminotransferase.

Role of tyrosine 65 in the mechanism of serine hydroxymethyltransferase.

Crystal structures of human and rabbit cytosolic serine hydroxymethyltransferase have shown that Tyr65 is likely to be a key residue in the mechanism of the enzyme. In the ternary complex of Escherichia coli serine hydroxymethyltransferase with glycine and 5-formyltetrahydrofolate, the hydroxyl of Tyr65 is one of four enzyme side chains within hydrogen-bonding distance of the carboxylate group of the substrate glycine. To probe the role of Tyr65 it was changed by site-directed mutagenesis to Phe65. The three-dimensional structure of the Y65F site mutant was determined and shown to be isomorphous with the wild-type enzyme except for the missing Tyr hydroxyl group. The kinetic properties of this mutant enzyme in catalyzing reactions with serine, glycine, allothreonine, D- and L-alanine, and 5,10-methenyltetrahydrofolate substrates were determined. The properties of the enzyme with D- and L-alanine, glycine in the absence of tetrahydrofolate, and 5, 10-methenyltetrahydrofolate were not significantly changed. However, catalytic activity was greatly decreased for serine and allothreonine cleavage and for the solvent alpha-proton exchange of glycine in the presence of tetrahydrofolate. The decreased catalytic activity for these reactions could be explained by a greater than 2 orders of magnitude increase in affinity of Y65F mutant serine hydroxymethyltransferase for these amino acids bound as the external aldimine. These data are consistent with a role for the Tyr65 hydroxyl group in the conversion of a closed active site to an open structure.

Vitamin B-6 Deficiency in Rats Reduces Hepatic Serine Hydroxymethyltransferase and Cystathionine β-Synthase Activities and Rates of In Vivo Protein Turnover, Homocysteine Remethylation and Transsulfuration

Vitamin B-6 deficiency causes mild elevation in plasma homocysteine, but the mechanism has not been clearly established. Serine is a substrate in one-carbon metabolism and in the transsulfuration pathway of homocysteine catabolism, and pyridoxal phosphate (PLP) plays a key role as coenzyme for serine hydroxymethyltransferase (SHMT) and enzymes of transsulfuration. In this study we used [(2)H(3)]serine as a primary tracer to examine the remethylation pathway in adequately nourished and vitamin B-6-deficient rats [7 and 0.1 mg pyridoxine (PN)/kg diet]. [(2)H(3)]Leucine and [1-(13)C]methionine were also used to examine turnover of protein and methionine pools, respectively. All tracers were injected intraperitoneally as a bolus dose, and then rats were killed (n = 4/time point) after 30, 60 and 120 min. Rats fed the low-PN diet had significantly lower growth and plasma and liver PLP concentrations, reduced liver SHMT activity, greater plasma and liver total homocysteine concentration, and reduced liver S-adenosylmethionine concentration. Hepatic and whole body protein turnover were reduced in vitamin B-6-deficient rats as evidenced by greater isotopic enrichment of [(2)H(3)]leucine. Hepatic [(2)H(2)]methionine production from [(2)H(3)]serine via cytosolic SHMT and the remethylation pathway was reduced by 80.6% in vitamin B-6 deficiency. The deficiency did not significantly reduce hepatic cystathionine-beta-synthase activity, and in vivo hepatic transsulfuration flux shown by production of [(2)H(3)]cysteine from the [(2)H(3)]serine increased over twofold. In contrast, plasma appearance of [(2)H(3)]cysteine was decreased by 89% in vitamin B-6 deficiency. The rate of hepatic homocysteine production shown by the ratio of [1-(13)C]homocysteine/[1-(13)C]methionine areas under enrichment vs. time curves was not affected by vitamin B-6 deficiency. Overall, these results indicate that vitamin B-6 deficiency substantially affects one-carbon metabolism by impairing both methyl group production for homocysteine remethylation and flux through whole-body transsulfuration.