Crystal structure at 2.4 A resolution of E. coli serine hydroxymethyltransferase in complex with glycine substrate and 5-formyl tetrahydrofolate

Serine hydroxymethyltransferase (EC 2.1.2.1), a member of the alpha-class of pyridoxal phosphate enzymes, catalyzes the reversible interconversion of serine and glycine, changing the chemical bonding at the C(alpha)-C(beta) bond of the serine side-chain mediated by the pyridoxal phosphate cofactor. Scission of the C(alpha)-C(beta) bond of serine substrate produces a glycine product and most likely formaldehyde, which reacts without dissociation with tetrahydropteroylglutamate cofactor. Crystal structures of the human and rabbit cytosolic serine hydroxymethyltransferases (SHMT) confirmed their close similarity in tertiary and dimeric subunit structure to each other and to aspartate aminotransferase, the archetypal alpha-class pyridoxal 5'-phosphate enzyme. We describe here the structure at 2.4 A resolution of Escherichia coli serine hydroxymethyltransferase in ternary complex with glycine and 5-formyl tetrahydropteroylglutamate, refined to an R-factor value of 17.4 % and R(free) value of 19.6 %. This structure reveals the interactions of both cofactors and glycine substrate with the enzyme. Comparison with the E. coli aspartate aminotransferase structure shows the distinctions in sequence and structure which define the folate cofactor binding site in serine hydroxymethyltransferase and the differences in orientation of the amino terminal arm, the evolution of which was necessary for elaboration of the folate binding site. Comparison with the unliganded rabbit cytosolic serine hydroxymethyltransferase structure identifies changes in the conformation of the enzyme, similar to those observed in aspartate aminotransferase, that probably accompany the binding of substrate. The tetrameric quaternary structure of liganded E. coli serine hydroxymethyltransferase also differs in symmetry and relative disposition of the functional tight dimers from that of the unliganded eukaryotic enzymes. SHMT tetramers have surface charge distributions which suggest distinctions in folate binding between eukaryotic and E. coli enzymes. The structure of the E. coli ternary complex provides the basis for a thorough investigation of its mechanism through characterization and structure determination of site mutants.     

l-Threonine aldolase, serine hydroxymethyltransferase and fungal alanine racemase. A subgroup of strictly related enzymes specialized for different functions.

Serine hydroxymethyltransferase (SHMT) is a member of the fold type I family of vitamin B6-dependent enzymes, a group of evolutionarily related proteins that share the same overall fold. The reaction catalysed by SHMT, the transfer of Cbeta of serine to tetrahydropteroylglutamate (H4PteGlu), represents in the cell an important link between the breakdown of amino acids and the metabolism of folates. In the absence of H4PteGlu and when presented with appropriate substrate analogues, SHMT shows a broad range of reaction specificity, being able to catalyse at appreciable rates retroaldol cleavage, racemase, aminotransferase and decarboxylase reactions. This apparent lack of specificity is probably a consequence of the particular catalytic apparatus evolved by SHMT. An interesting question is whether other fold type I members that normally catalyse the reactions which for SHMT could be considered as 'forced errors', may be close relatives of this enzyme and have a catalytic apparatus with the same basic features. As shown in this study, l-threonine aldolase from Escherichia coli is able to catalyse the same range of reactions catalysed by SHMT, with the exception of the serine hydroxymethyltransferase reaction. This observation strongly suggests that SHMT and l-threonine aldolase are closely related enzymes specialized for different functions. An evolutionary analysis of the fold type I enzymes revealed that SHMT and l-threonine aldolase may actually belong to a subgroup of closely related proteins; fungal alanine racemase, an extremely close relative of l-threonine aldolase, also appears to be a member of the same subgroup. The construction of three-dimensional homology models of l-threonine aldolase from E. coli and alanine racemase from Cochliobolus carbonum, and their comparison with the SHMT crystal structure, indicated how the tetrahydrofolate binding site might have evolved and offered a starting point for further investigations.     

Serine hydroxymethyltransferase: a model enzyme for mechanistic, structural, and evolutionary studies

Serine hydroxymethyltransferase is a ubiquitous representative of the family of fold type I, pyridoxal 5'-phosphate-dependent enzymes. The reaction catalyzed by this enzyme, the reversible transfer of the Cβ of serine to tetrahydropteroylglutamate, represents a link between amino acid and folates metabolism and operates as a major source of one-carbon units for several essential biosynthetic processes. Serine hydroxymethyltransferase has been intensively investigated because of the interest aroused by the complex mechanism of the hydroxymethyltransferase reaction and its broad substrate and reaction specificity. Although the increasing availability of crystallographic data and the characterization of several site-specific mutants helped in understanding previous functional and structural studies, they also represent the starting point of novel investigations. This review will focus on recently highlighted catalytic, structural, and evolutionary aspects of serine hydroxymethyltransferase. This article is part of a Special Issue entitled: Pyridoxal phosphate Enzymology.     

Purification and properties of serine hydroxymethyltransferase from Sulfolobus solfataricus.

Serine hydroxymethyltransferase (SHMT) catalyzes the reversible cleavage of serine to glycine with the transfer of the one-carbon group to tetrahydrofolate to form 5,10-methylenetetrahydrofolate. No SHMT has been purified from a nonmethanogenic Archaea strain, in part because this group of organisms uses modified folates as the one-carbon acceptor. These modified folates are not readily available for use in assays for SHMT activity. This report describes the purification and characterization of SHMT from the thermophilic organism Sulfolobus solfataricus. The exchange of the alpha-proton of glycine with solvent protons in the absence of the modified folate was used as the activity assay. The purified protein catalyzes the synthesis of serine from glycine and a synthetic derivative of a fragment of the natural modified folate found in S. solfataricus. Replacement of the modified folate with tetrahydrofolate did not support serine synthesis. In addition, this SHMT also catalyzed the cleavage of both allo-threonine and beta-phenylserine in the absence of the modified folate. The cleavage of these two amino acids in the absence of tetrahydrofolate is a property of other characterized SHMTs. The enzyme contains covalently bound pyridoxal phosphate. Sequences of three peptides showed significant similarity with those of peptides of SHMTs from two methanogens.     

In vitro conversion of formate to serine: effect of tetrahydropteroylpolyglutamates and serine hydroxymethyltransferase on the rate of 10-formyltetrahydrofolate synthetase

Serine hydroxymethyltransferase and C1-tetrahydrofolate synthase catalyze four reactions which convert formate and glycine to serine. The one-carbon carrier in these reactions if tetrahydropteroylglutamate which is regenerated in the coupled reaction and thus can be used in catalytic concentrations with respect to serine synthesis. The rate of serine synthesis is followed by the oxidation of NADPH during reduction of the intermediate 5,10-methenyltetrahydropteroylglutamate. Km values for the substrates of cytosolic serine hydroxymethyltransferase and the 10-formyltetrahydrofolate synthetase activity of the trifunctional enzyme C1-tetrahydrofolate synthase were determined. This included the values for the polyglutamate forms of tetrahydropteroylglutamate containing from one to six glutamate residues. The results suggest that the synthetase active site binds the polyglutamate forms of the coenzyme synergistically with respect to formate and ATP. Using saturating levels of all substrates, the kcat values for the serine hydroxymethyltransferase and 10-formyltetrahydrofolate synthetase activities were also determined. The synthetase reaction is the rate-determining step in the conversion of formate to serine. The effect of glutamate chain length and the concentration of serine hydroxymethyltransferase were studied with respect to the rate of serine formation. Tetrahydropteroylmonoglutamate gave slower than expected rates which is attributed to its inhibition of the reduction of the intermediate 5,10-methenyltetrahydropteroylglutamate. This inhibition was not a factor with the di- through hexaglutamate forms of the coenzyme. The addition of an excess of serine hydroxymethyltransferase was predicted to lower the rate of the formation of serine by lowering the concentration of free coenzyme in the assay. However, activation of the rate was observed which was at least 2-fold greater than the predicted rate. This increase in predicted rate appears to result from an interaction between C1-tetrahydrofolate synthase and serine hydroxymethyltransferase. The in vivo concentrations of serine hydroxymethyltransferase and C1-tetrahydrofolate synthase in rabbit liver were determined.     

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.     

Role of Lys-226 in the catalytic mechanism of Bacillus stearothermophilus serine hydroxymethyltransferase--crystal structure and kinetic studies

Serine hydroxymethyltransferase (SHMT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme catalyzes the reversible conversion of l-Ser and tetrahydropteroylglutamate (H(4)PteGlu) to Gly and 5,10-methylene tetrahydropteroylglutamate (CH(2)-H(4)PteGlu). Biochemical and structural studies on this enzyme have implicated several residues in the catalytic mechanism, one of them being the active site lysine, which anchors PLP. It has been proposed that this residue is crucial for product expulsion. However, in other PLP-dependent enzymes, the corresponding residue has been implicated in the proton abstraction step of catalysis. In the present investigation, Lys-226 of Bacillus stearothermophilus SHMT (bsSHMT) was mutated to Met and Gln to evaluate the role of this residue in catalysis. The mutant enzymes contained 1 mol of PLP per mol of subunit suggesting that Schiff base formation with lysine is not essential for PLP binding. The 3D structure of the mutant enzymes revealed that PLP was bound at the active site in an orientation different from that of the wild-type enzyme. In the presence of substrate, the PLP ring was in an orientation superimposable with that of the external aldimine complex of wild-type enzyme. However, the mutant enzymes were inactive, and the kinetic analysis of the different steps of catalysis revealed that there was a drastic reduction in the rate of formation of the quinonoid intermediate. Analysis of these results along with the crystal structures suggested that K-226 is responsible for flipping of PLP from one orientation to another which is crucial for H(4)PteGlu-dependent Calpha-Cbeta bond cleavage of l-Ser.     

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 kcat and Km 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.     

Conformational transitions driven by pyridoxal‐5′‐phosphate uptake in the psychrophilic serine hydroxymethyltransferase from Psychromonas ingrahamii

Serine hydroxymethyltransferase (SHMT) is a pyridoxal‐5′‐phosphate (PLP)‐dependent enzyme belonging to the fold type I superfamily, which catalyzes in vivo the reversible conversion of l ‐serine and tetrahydropteroylglutamate (H₄PteGlu) to glycine and 5,10‐methylenetetrahydropteroylglutamate (5,10‐CH₂‐H₄PteGlu). The SHMT from the psychrophilic bacterium Psychromonas ingrahamii (piSHMT) had been recently purified and characterized. This enzyme was shown to display catalytic and stability properties typical of psychrophilic enzymes, namely high catalytic activity at low temperature and thermolability. To gain deeper insights into the structure–function relationship of piSHMT, the three‐dimensional structure of its apo form was determined by X‐ray crystallography. Homology modeling techniques were applied to build a model of the piSHMT holo form. Comparison of the two forms unraveled the conformation modifications that take place when the apo enzyme binds its cofactor. Our results show that the apo form is in an “open” conformation and possesses four (or five, in chain A) disordered loops whose electron density is not visible by X‐ray crystallography. These loops contain residues that interact with the PLP cofactor and three of them are localized in the major domain that, along with the small domain, constitutes the single subunit of the SHMT homodimer. Cofactor binding triggers a rearrangement of the small domain that moves toward the large domain and screens the PLP binding site at the solvent side. Comparison to the mesophilic apo SHMT from Salmonella typhimurium suggests that the backbone conformational changes are wider in psychrophilic SHMT. Proteins 2014; 82:2831–2841. © 2014 Wiley Periodicals, Inc.     

Structure of a murine cytoplasmic serine hydroxymethyltransferase quinonoid ternary complex: evidence for asymmetric obligate dimers

Serine hydroxymethyltransferase (SHMT) is a pyridoxal phosphate-dependent enzyme that catalyzes the reversible conversion of serine and tetrahydrofolate to glycine and methylenetetrahydrofolate. This reaction generates single carbon units for purine, thymidine, and methionine biosynthesis. The enzyme is a homotetramer comprising two obligate dimers and four pyridoxal phosphate-bound active sites. The mammalian enzyme is present in cells in both catalytically active and inactive forms. The inactive form is a ternary complex that results from the binding of glycine and 5-formyltetrahydrofolate polyglutamate, a slow tight-binding inhibitor. The crystal structure of a close analogue of the inactive form of murine cytoplasmic SHMT (cSHMT), lacking only the polyglutamate tail of the inhibitor, has been determined to 2.9 A resolution. This first structure of a ligand-bound mammalian SHMT allows identification of amino acid residues involved in substrate binding and catalysis. It also reveals that the two obligate dimers making up a tetramer are not equivalent; one can be described as "tight-binding" and the other as "loose-binding" for folate. Both active sites of the tight-binding dimer are occupied by 5-formyltetrahydrofolate (5-formylTHF), whose N5-formyl carbon is within 4 A of the glycine alpha-carbon of the glycine-pyridoxal phosphate complex; the complex appears to be primarily in its quinonoid form. In the loose-binding dimer, 5-formylTHF is present in only one of the active sites, and its N5-formyl carbon is 5 A from the glycine alpha-carbon. The pyridoxal phosphates appear to be primarily present as geminal diamine complexes, with bonds to both glycine and the active site lysine. This structure suggests that only two of the four catalytic sites on SHMT are catalytically competent and that the cSHMT-glycine-5-formylTHF ternary complex is an intermediate state analogue of the catalytic complex associated with serine and glycine interconversion.     

Escherichia coli K12 mutants defective in the glycine cleavage enzyme system

Two routes of one-carbon biosynthesis have been described in Escherichia coli K12. One is from serine via the serine hydroxymethyltransferase (SHMT) reaction, and the other is from glycine via the glycine cleavage (GCV) enzyme system. To isolate mutants deficient in the GCV pathway, we used a selection procedure that is based on the assumption that loss of this enzyme system in strains blocked in serine biosynthesis results in their inability to use glycine as a serine source. Mutants were accordingly isolated that grow with a serine supplement, but not with a glycine supplement. Enzyme assays demonstrated that three independently isolated mutants have no detectable GCV enzyme activity. The absence of a functional GCV pathway results in the excretion of glycine, but has no affect on the cell's primary source of one-carbon units, the SHMT reaction. The new mutations, designated gcv, were mapped between the serA and lysA genes on the E. coli chromosome.     

Divergent adaptation of tRNA recognition by Methanococcus jannaschii prolyl-tRNA synthetase

Analysis of prolyl-tRNA synthetase (ProRS) across all three taxonomic domains (Eubacteria, Eucarya, and Archaea) reveals that the sequences are divided into two distinct groups. Recent studies show that Escherichia coli ProRS, a member of the "prokaryotic-like" group, recognizes specific tRNA bases at both the acceptor and anticodon ends, whereas human ProRS, a member of the "eukaryotic-like" group, recognizes nucleotide bases primarily in the anticodon. The archaeal Methanococcus jannaschii ProRS is a member of the eukaryotic-like group, although its tRNA(Pro) possesses prokaryotic features in the acceptor stem. We show here that, in some respects, recognition of tRNA(Pro) by M. jannaschii ProRS parallels that of human, with a strong emphasis on the anticodon and only weak recognition of the acceptor stem. However, our data also indicate differences in the details of the anticodon recognition between these two eukaryotic-like synthetases. Although the human enzyme places a stronger emphasis on G35, the M. jannaschii enzyme places a stronger emphasis on G36, a feature that is shared by E. coli ProRS. These results, interpreted in the context of an extensive sequence alignment, provide evidence of divergent adaptation by M. jannaschii ProRS; recognition of the tRNA acceptor end is eukaryotic-like, whereas the details of the anticodon recognition are prokaryotic-like. This divergence may be a reflection of the unusual dual function of this enzyme, which catalyzes specific aminoacylation with proline as well as with cysteine.     

Crystal structure of binary and ternary complexes of serine hydroxymethyltransferase from Bacillus stearothermophilus: insights into the catalytic mechanism

Serine hydroxymethyltransferase (SHMT), a member of the alpha-class of pyridoxal phosphate-dependent enzymes, catalyzes the reversible conversion of serine to glycine and tetrahydrofolate to 5,10-methylene tetrahydrofolate. We present here the crystal structures of the native enzyme and its complexes with serine, glycine, glycine, and 5-formyl tetrahydrofolate (FTHF) from Bacillus stearothermophilus. The first structure of the serine-bound form of SHMT allows identification of residues involved in serine binding and catalysis. The SHMT-serine complex does not show any significant conformational change compared with the native enzyme, contrary to that expected for a conversion from an "open" to "closed" form of the enzyme. However, the ternary complex with FTHF and glycine shows the reported conformational changes. In contrast to the Escherichia coli enzyme, this complex shows asymmetric binding of the FTHF to the two monomers within the dimer in a way similar to the murine SHMT. Comparison of the ternary complex with the native enzyme reveals the structural basis for the conformational change and asymmetric binding of FTHF. The four structures presented here correspond to the various reaction intermediates of the catalytic pathway and provide evidence for a direct displacement mechanism for the hydroxymethyl transfer rather than a retroaldol cleavage.     

Cytoplasmic serine hydroxymethyltransferase regulates the metabolic partitioning of methylenetetrahydrofolate but is not essential in mice

The hydroxymethyl group of serine is a primary source of tetrahydrofolate (THF)-activated one-carbon units that are required for the synthesis of purines and thymidylate and for S-adenosylmethionine (AdoMet)-dependent methylation reactions. Serine hydroxymethyltransferase (SHMT) catalyzes the reversible and THF-dependent conversion of serine to glycine and 5,10-methylene-THF. SHMT is present in eukaryotic cells as mitochondrial SHMT and cytoplasmic (cSHMT) isozymes that are encoded by distinct genes. In this study, the essentiality of cSHMT-derived THF-activated one-carbons was investigated by gene disruption in the mouse germ line. Mice lacking cSHMT are viable and fertile, demonstrating that cSHMT is not an essential source of THF-activated one-carbon units. cSHMT-deficient mice exhibit altered hepatic AdoMet levels and uracil content in DNA, validating previous in vitro studies that indicated this enzyme regulates the partitioning of methylenetetrahydrofolate between the thymidylate and homocysteine remethylation pathways. This study suggests that mitochondrial SHMT-derived one-carbon units are essential for folate-mediated one-carbon metabolism in the cytoplasm.     

Design,docking studies and molecular dynamics of new potential selective inhibitors of Plasmodium falciparum serine hydroxymethyltransferase

In this paper, former studies on the interactions of the natural substrate and potential inhibitors of Plasmodium falciparum serine hydroxymethyltransferase (PfSHMT) were used to design five new potential selective inhibitors to this enzyme. Results of the docking energies calculations of these structures inside the active sites of PfSHMT and human SHMT were used to select a more suitable structure as a potential selective inhibitor to PfSHMT. Further molecular dynamics studies of this molecule and 5-formyl-6-hydrofolic acid (natural substrate) docked inside these enzymes' active sites revealed important features for additional refinements of this structure and also additional residues in the PfSHMT active site to be considered further for designing selective inhibitors.     

Factors affecting the biosynthesis of L-tryptophan by genetically modified strains of Escherichia coli

Derivatives of Escherichia coli strain W3110 with increased tryptophan synthase (TS) activity were constructed. The biosynthesis of serine was shown to limit tryptophan production in minimal medium with indole as precursor. In the presence of serine and indole we obtained a correlation between the specific activity of TS and the specific productivity (qp) of tryptophan. Supplementation of the growth medium with glycine enhanced qp two-fold. In a strain with high serine hydroxymethyltransferase (SHMT) activity no such increase of tryptophan productivity was observed, although crude extracts from these cells were shown to produce tryptophan with indole, one-carbon units and glycine as precursors. Growth of the strain with high SHMT activity was inhibited in a medium with high glycine concentration. This inhibition could not be released by addition of isoleucine and valine. In a buffer system with permeabilized cells high in specific TS and SHMT activities we did not obtain any tryptophan production in presence of indole, glycine, one-carbon units and cofactors. On the other hand, in a buffer system with indole and serine as precursors we obtained high qp of tryptophan [13.3 g tryptophan (g dry wt cells)-1 h-1], which was correlated to the TS specific activity.     

Cloning and structural analysis of Mycobacterium leprae serine hydroxymethyltransferase

Serine hydroxymethyltransferase (SHMT) plays a key role in cell physiology as it participates in the different interconversion pathway of folate coenzymes, provides almost exclusively folate one carbon fragments for the biosynthesis of a variety of end products. For the first time, Mycobacterium leprae glyA gene, encodes the enzyme serine hydroxymethyltransferase, has been cloned in Escherichia coli, over-expressed and purified the protein product (mlSHMT) for folding and stability studies under various denaturating conditions. The recombinant mlSHMT exists as homo-dimer of molecular mass about 90 kDa under physiological conditions . The studies on catalytic properties of mlSHMT show that the enzyme catalyzes the H(4)-folate dependent retro-aldol cleavage of L-serine, however, D-alanine dependent transaminase activity was absent in the enzyme. Further analysis of the enzyme kinetics for hydroxymethyltransferase reaction for mlSHMT demonstrates a comparable K(m) value for L-serine to SHMTs from other sources but significantly lower catalytic efficiency (k(cat)/K(m)). The mlSHMT is resistant to alkaline denaturation and exist as apo-dimer up to pH 10.5. Urea and guanidinium chloride induces dissociation of mlSHMT dimer into monomer at low denaturant concentrations, and leads to loss of enzymatic activity.     

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.     

Crystal structure of rabbit cytosolic serine hydroxymethyltransferase at 2.8 A resolution: mechanistic implications.

Serine hydroxymethyltransferase (SHMT) catalyzes the reversible cleavage of serine to form glycine and single carbon groups that are essential for many biosynthetic pathways. SHMT requires both pyridoxal phosphate (PLP) and tetrahydropteroylpolyglutamate (H4PteGlun) as cofactors, the latter as a carrier of the single carbon group. We describe here the crystal structure at 2.8 A resolution of rabbit cytosolic SHMT (rcSHMT) in two forms: one with the PLP covalently bound as an aldimine to the Nepsilon-amino group of the active site lysine and the other with the aldimine reduced to a secondary amine. The rcSHMT structure closely resembles the structure of human SHMT, confirming its similarity to the alpha-class of PLP enzymes. The structures reported here further permit identification of changes in the PLP group that accompany formation of the geminal diamine complex, the first intermediate in the reaction pathway. On the basis of the current mechanism derived from solution studies and the properties of site mutants, we are able to model the binding of both the serine substrate and the H4PteGlun cofactor. This model explains the properties of several site mutants of SHMT and offers testable hypotheses for a more detailed mechanism of this enzyme.     

Role of proline residues in the folding of serine hydroxymethyltransferase

Previous studies on the folding mechanism of Escherichia coli serine hydroxymethyltransferase (SHMT) showed that the final rate determining folding step was from an intermediate that contained two fully folded domains with N-terminal segments of approximately 55 residues and interdomain segments of approximately 50 residues that were still solvent exposed and subject to proteolysis. The interdomain segment contains 3 Pro residues near its N terminus and 2 Pro residues near its C terminus. The 5 Pro residues were each mutated to both a Gly and Ala residue, and each mutant SHMT was purified and characterized with respect to kinetic properties, stability, secondary structure, and folding mechanism. The results showed that Pro214 and Pro218 near the N terminus of the interdomain segment are not critical for folding, stability, or activity. The P216A mutant also retained most of the characteristics of the native enzyme, but its folding rate was altered. However, the P216G mutant was severely compromised in folding into a catalytically competent enzyme. Mutation of both Pro258 and Pro264 had altered folding kinetics and resulted in enzymes that expressed little catalytic activity. The Phe257-Pro258 bond is cis in its configuration, and the P258A mutant SHMT showed reduced thermal stability. Pro216, Pro258, and Pro264 are conserved in all 53 known sequences of this enzyme. The results are discussed in terms of the role of each Pro residue in maintaining the structure and function of SHMT and a possible role in pyridoxal 5'-phosphate addition to the apo-enzyme.