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Verne Schirch - One of the best experts on this subject based on the ideXlab platform.
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Structure-based mechanism for early PLP-mediated steps of rabbit cytosolic Serine Hydroxymethyltransferase reaction
BioMed research international, 2013Co-Authors: Martino L. Di Salvo, Verne Schirch, Roberto Contestabile, G. Kazanina, J. Neel Scarsdale, H. Tonie WrightAbstract:Serine Hydroxymethyltransferase catalyzes the reversible interconversion of L-Serine and glycine with transfer of one-carbon groups to and from tetrahydrofolate. Active site residue Thr254 is known to be involved in the transaldimination reaction, a crucial step in the catalytic mechanism of all pyridoxal 5′-phosphate- (PLP-) dependent enzymes, which determines binding of substrates and release of products. In order to better understand the role of Thr254, we have expressed, characterized, and determined the crystal structures of rabbit cytosolic Serine Hydroxymethyltransferase T254A and T254C mutant forms, in the absence and presence of substrates. These mutants accumulate a kinetically stable gem-diamine intermediate, and their crystal structures show differences in the active site with respect to wild type. The kinetic and crystallographic data acquired with mutant enzymes permit us to infer that conversion of gem-diamine to external aldimine is significantly slowed because intermediates are trapped into an anomalous position by a misorientation of the PLP ring, and a new energy barrier hampers the transaldimination reaction. This barrier likely arises from the loss of the stabilizing hydrogen bond between the hydroxymethyl group of Thr254 and the e-amino group of active site Lys257, which stabilizes the external aldimine intermediate in wild type SHMTs.
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Serine Hydroxymethyltransferase revisited.
Current Opinion in Chemical Biology, 2005Co-Authors: Verne Schirch, Doletha M. E. SzebenyiAbstract:Recent structural data and the properties of several active site mutants of Serine Hydroxymethyltransferase have resolved some key questions concerning the catalytic mechanism and broad substrate specificity of this enzyme. In the tetrahydrofolate-dependent conversion of Serine to glycine, an early proposed mechanism involved a retroaldol cleavage and a formaldehyde intermediate, while a more recent suggestion posits a direct nucleophilic displacement of the Serine hydroxyl by N5 of tetrahydrofolate, without creation of free formaldehyde. Geometric and chemical difficulties with both options led to a new proposal, a modified retroaldol mechanism in which N5 of tetrahydrofolate makes a nucleophilic attack on Serine C3 leading to breakage of the C3–C2-bond of Serine rather than the C3-hydroxyl bond. Molecular modeling revealed how a variety of substrates could be accommodated in the folate-independent cleavage of 3-hydroxyamino acids and shed light on the mechanism of this reaction.
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Role of tyrosine 65 in the mechanism of Serine Hydroxymethyltransferase.
Biochemistry, 2000Co-Authors: Roberto Contestabile, Sebastiana Angelaccio, Francesco Bossa, G. Kazanina, H.t. Wright, N. Scarsdale, Verne SchirchAbstract: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. ...
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Crystal structure at 2.4 A resolution of E. coli Serine Hydroxymethyltransferase in complex with glycine substrate and 5-formyl tetrahydrofolate.
Journal of molecular biology, 2000Co-Authors: J.n. Scarsdale, Verne Schirch, G. Kazanina, S. Radaev, H.t. WrightAbstract: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.
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crystal structure at 2 4 a resolution of e coli Serine Hydroxymethyltransferase in complex with glycine substrate and 5 formyl tetrahydrofolate
Journal of Molecular Biology, 2000Co-Authors: J.n. Scarsdale, Verne Schirch, G. Kazanina, S. Radaev, H.t. WrightAbstract:Abstract Serine Hydroxymethyltransferase (EC 2.1.2.1), a member of the α-class of pyridoxal phosphate enzymes, catalyzes the reversible interconversion of Serine and glycine, changing the chemical bonding at the C α -C β bond of the Serine side-chain mediated by the pyridoxal phosphate cofactor. Scission of the C α -C β 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 α-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.
Handanahal S. Savithri - One of the best experts on this subject based on the ideXlab platform.
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Role of pro-297 in the catalytic mechanism of sheep liver Serine Hydroxymethyltransferase.
Biochemical Journal, 2000Co-Authors: Rashmi Talwar, Vijayapandian Leelavathy, Jala V. Krishna Rao, Naropantul Appaji Rao, Handanahal S. SavithriAbstract:Serine Hydroxymethyltransferase belongs to the a 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 c-carboxy group of aspartate. In an attempt to change the reaction speciAEcity 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 signiAE-cantly (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 speciAEcity, unlike the similarly positioned Arg-292 in aspartate aminotransferase.
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Molecular organization, catalytic mechanism and function of Serine Hydroxymethyltransferase — a potential target for cancer chemotherapy
The international journal of biochemistry & cell biology, 2000Co-Authors: N. Appaji Rao, Rashmi Talwar, Handanahal S. SavithriAbstract:Serine Hydroxymethyltransferase, a pyridoxal-5′-phosphate dependent enzyme, catalyzes the retro-aldol cleavage of Serine to yield glycine and the hydroxymethyl group is transferred to 5,6,7,8-tetrahydrofolate to generate 5,10-methylene-$H_4$-folate. The enzyme plays a pivotal role in channeling metabolites between amino acid and nucleotide metabolism. Dihydrofolate reductase and thymidylate synthase have been favorite targets for the development of anticancer drugs. However, development of resistance to drugs, due to a variety of reasons, has necessitated the identification of alternate targets for cancer chemotherapy and Serine Hydroxymethyltransferase is one such potential target. A detailed study of the kinetics of interaction of Serine and folate analogs with this enzyme revealed several unique features that can be exploited for the design of new chemotherapeutic agents. The pathways for the reversible unfolding of the dimeric Escherichia coli and the tetrameric sheep liver enzyme, although different, revealed a requirement for the cofactor in the final step for generating an active enzyme. The gly A gene of Escherichia coli has been shown to code for this enzyme. Analysis of available gene sequences indicate that Serine Hydroxymethyltransferase is one of the most highly conserved proteins. The isolation of the cDNA clones for the enzyme and their overexpression in heterologous systems has enabled the probing of the molecular mechanisms of catalysis and the role of lysine, arginine and histidine in cofactor, substrate(s) binding and in maintaining the structure of the protein. Recently, the three-dimensional structure of the human liver Serine Hydroxymethyltransferase has been published. This, along with the information already available, provides a framework for the rational design of drugs targeted specifically towards this enzyme.
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A change in reaction specificity of sheep liver Serine Hydroxymethyltransferase. Induction of NADH oxidation upon mutation of His230 to Tyr.
European journal of biochemistry, 2000Co-Authors: Rashmi Talwar, N. Appaji Rao, Handanahal S. SavithriAbstract:Both Serine Hydroxymethyltransferase and aspartate aminotransferase belong to the α-class of pyridoxal-5′-phosphate (pyridoxalP)-dependent enzymes but exhibit different reaction and substrate specificities. A comparison of the X-ray structure of these two enzymes reveals that their active sites are nearly superimposable. In an attempt to change the reaction specificity of Serine Hydroxymethyltransferase to a transaminase, His 230 was mutated to Tyr which is the equivalent residue in aspartate aminotransferase. Surprisingly, the H230Y mutant was found to catalyze oxidation of NADH in an enzyme concentration dependent manner instead of utilizing l-aspartate as a substrate. The NADH oxidation could be linked to oxygen consumption or reduction of nitrobluetetrazolium. The reaction was inhibited by radical scavengers like superoxide dismutase and d-mannitol. The Km and kcat values for the reaction of the enzyme with NADH were 74 µm and 5.2 × 10−3 s−1, respectively. This oxidation was not observed with either the wild type Serine Hydroxymethyltransferase or H230A, H230F or H230N mutants. Thus, mutation of H230 of sheep liver Serine Hydroxymethyltransferase to Tyr leads to induction of an NADH oxidation activity implying that tyrosyl radicals may be mediating the reaction.
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The primary structure of sheep liver cytosolic Serine Hydroxymethyltransferase and an analysis of the evolutionary relationships among Serine Hydroxymethyltransferases.
Biochimica et biophysica acta, 1994Co-Authors: Rajagopalan Usha, Handanahal S. Savithri, N. Appaji RaoAbstract:The complete amino-acid sequence of sheep liver cytosolic Serine Hydroxymethyltransferase was determined from an analysis of tryptic, chymotryptic, CNBr and hydroxylamine peptides. Each subunit of sheep liver Serine Hydroxymethyltransferase consisted of 483 amin-acid residues. A comparison of this sequence with 8 other Serine Hydroxymethyltransferases revealed that a possible gene duplication event could have occurred after the divergence of animals and fungi. This analysis also showed independent duplication of SHMT genes in Neurospora crassa. At the secondary structural level, all the Serine Hydroxymethyltransferases belong to the α/β category of proteins. The predicted secondary structure of sheep liver Serine Hydroxymethyltransferase was similar to that of the observed structure of tryptophan synthase, another pyridoxal 5′-phosphate containing enzyme, suggesting that sheep liver Serine Hydroxymethyltransferase might have a similar pyridoxal 5′-phosphate binding domain. In addition, a conserved glycinerich region, G L Q G G P, was identified in all the Serine Hydroxymethyltransferases and could be important in pyridoxal 5′-phosphate binding. A comparison of the cytosolic Serine Hydroxymethyltransferases from rabbit and sheep liver with other proteins sequenced from both these sources showed that Serine Hydroxymethyltransferase was a highly conserved protein. In was slightly less conserved than cytochrome c but better conserved than myoglobin, both of which are well known evolutionary markers. C67 and C203 were specifically protected by pyridoxal 5′-phosphate against modification with [14C]iodoacetic acid, while C247 and C261 were buried in the native Serine Hydroxymethyltransferase. However, the cysteines are not conserved among the various Serine Hydroxymethyltransferases. The exact role of the cysteines in the reaction catalyzed by Serine Hydroxymethyltransferase remains to be elucidated.
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Arginine residues involved in binding of tetrahydrofolate to sheep liver Serine Hydroxymethyltransferase.
Journal of Biological Chemistry, 1992Co-Authors: R Usha, Handanahal S. SavithriAbstract:Abstract The arginine residue(s) necessary for tetrahydrofolate binding to sheep liver Serine Hydroxymethyltransferase were located by phenylglyoxal modification. The incorporation of [7-14C]phenylglyoxal indicated that 2 arginine residues were modified per subunit of the enzyme and the modification of these residues was prevented by tetrahydrofolate. In order to locate the sites of phenylglyoxal modification, the enzyme was reacted in the presence and absence of tetrahydrofolate using unlabeled and radioactive phenylglyoxal, respectively. The labeled phenylglyoxal-treated enzyme was digested with trypsin, and the radiolabeled peptides were purified by high-performance liquid chromatography on reversed-phase columns. Sequencing the tryptic peptides indicated that Arg-269 and Arg-462 were the sites of phenylglyoxal modification. Neither a spectrally discernible 495-nm intermediate (characteristic of the native enzyme when substrates are added) nor its enhancement by the addition of tetrahydrofolate, was observed with the phenylglyoxal-modified enzyme. There was no enhancement of the rate of the exchange of the alpha-proton of glycine upon addition of tetrahydrofolate to the modified enzyme as was observed with the native enzyme. These results demonstrate the requirement of specific arginine residues for the interaction of tetrahydrofolate with sheep liver Serine Hydroxymethyltransferase.
Roberto Contestabile - One of the best experts on this subject based on the ideXlab platform.
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Human Cytosolic and Mitochondrial Serine Hydroxymethyltransferase Isoforms in Comparison: Full Kinetic Characterization and Substrate Inhibition Properties.
Biochemistry, 2018Co-Authors: Angela Tramonti, Martino L. Di Salvo, Caterina Nardella, Anna Barile, Francesca Cutruzzolà, Roberto ContestabileAbstract:Serine Hydroxymethyltransferase (SHMT) catalyzes the reversible conversion of l-Serine and tetrahydrofolate into glycine and 5,10-methylenetetrahydrofolate. This enzyme, which plays a pivotal role in one-carbon metabolism, is involved in cancer metabolic reprogramming and is a recognized target of chemotherapy intervention. In humans, two isoforms of the enzyme exist, which are commonly termed cytosolic SHMT1 and mitochondrial SHMT2. Considerable attention has been paid to the structural, mechanistic, and metabolic features of these isozymes. On the other hand, a detailed comparison of their catalytic and regulatory properties is missing, although this aspect seems to be considerably important, considering that SHMT1 and SHMT2 reside in different cellular compartments, where they play distinct roles in folate metabolism. Here we performed a full kinetic characterization of the Serine Hydroxymethyltransferase reaction catalyzed by SHMT1 and SHMT2, with a focus on pH dependence and substrate inhibition. Our...
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Structure-based mechanism for early PLP-mediated steps of rabbit cytosolic Serine Hydroxymethyltransferase reaction
BioMed research international, 2013Co-Authors: Martino L. Di Salvo, Verne Schirch, Roberto Contestabile, G. Kazanina, J. Neel Scarsdale, H. Tonie WrightAbstract:Serine Hydroxymethyltransferase catalyzes the reversible interconversion of L-Serine and glycine with transfer of one-carbon groups to and from tetrahydrofolate. Active site residue Thr254 is known to be involved in the transaldimination reaction, a crucial step in the catalytic mechanism of all pyridoxal 5′-phosphate- (PLP-) dependent enzymes, which determines binding of substrates and release of products. In order to better understand the role of Thr254, we have expressed, characterized, and determined the crystal structures of rabbit cytosolic Serine Hydroxymethyltransferase T254A and T254C mutant forms, in the absence and presence of substrates. These mutants accumulate a kinetically stable gem-diamine intermediate, and their crystal structures show differences in the active site with respect to wild type. The kinetic and crystallographic data acquired with mutant enzymes permit us to infer that conversion of gem-diamine to external aldimine is significantly slowed because intermediates are trapped into an anomalous position by a misorientation of the PLP ring, and a new energy barrier hampers the transaldimination reaction. This barrier likely arises from the loss of the stabilizing hydrogen bond between the hydroxymethyl group of Thr254 and the e-amino group of active site Lys257, which stabilizes the external aldimine intermediate in wild type SHMTs.
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Glycine consumption and mitochondrial Serine Hydroxymethyltransferase in cancer cells: The heme connection
Medical hypotheses, 2013Co-Authors: Martino L. Di Salvo, Roberto Contestabile, Alessandro Paiardini, Bruno MarasAbstract: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 chemotherapeutic agents.
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Serine Hydroxymethyltransferase: A model enzyme for mechanistic, structural, and evolutionary studies
Biochimica et biophysica acta, 2010Co-Authors: Rita Florio, Mirella Vivoli, Martino L. Di Salvo, Roberto ContestabileAbstract:Abstract 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.
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role of a conserved active site cation π interaction in escherichia coli Serine Hydroxymethyltransferase
Biochemistry, 2009Co-Authors: Mirella Vivoli, Veronica Morea, Francesco Angelucci, Sebastiana Angelaccio, Martino L. Di Salvo, Andrea Ilari, Roberto ContestabileAbstract:Serine Hydroxymethyltransferase is a pyridoxal 5′-phosphate-dependent enzyme that catalyzes the interconversion of Serine and glycine using tetrahydropteroylglutamate as the one-carbon carrier. In all pyridoxal phosphate-dependent enzymes, amino acid substrates are bound and released through a transaldimination process, in which an internal aldimine and an external aldimine are interconverted via gem-diamine intermediates. Bioinformatic analyses of Serine Hydroxymethyltransferase sequences and structures showed the presence of two highly conserved residues, a tyrosine and an arginine, engaged in a cation−π interaction. In Escherichia coli Serine hydroxymethyltranferase, the hydroxyl group of this conserved tyrosine (Tyr55) is located in a position compatible with a role as hydrogen exchanger in the transaldimination reaction. Because of the location of Tyr55 at the active site, the enhancement of its acidic properties caused by the cation−π interaction with Arg235, and the hydrogen bonds established by it...
N. Appaji Rao - One of the best experts on this subject based on the ideXlab platform.
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Molecular organization, catalytic mechanism and function of Serine Hydroxymethyltransferase — a potential target for cancer chemotherapy
The international journal of biochemistry & cell biology, 2000Co-Authors: N. Appaji Rao, Rashmi Talwar, Handanahal S. SavithriAbstract:Serine Hydroxymethyltransferase, a pyridoxal-5′-phosphate dependent enzyme, catalyzes the retro-aldol cleavage of Serine to yield glycine and the hydroxymethyl group is transferred to 5,6,7,8-tetrahydrofolate to generate 5,10-methylene-$H_4$-folate. The enzyme plays a pivotal role in channeling metabolites between amino acid and nucleotide metabolism. Dihydrofolate reductase and thymidylate synthase have been favorite targets for the development of anticancer drugs. However, development of resistance to drugs, due to a variety of reasons, has necessitated the identification of alternate targets for cancer chemotherapy and Serine Hydroxymethyltransferase is one such potential target. A detailed study of the kinetics of interaction of Serine and folate analogs with this enzyme revealed several unique features that can be exploited for the design of new chemotherapeutic agents. The pathways for the reversible unfolding of the dimeric Escherichia coli and the tetrameric sheep liver enzyme, although different, revealed a requirement for the cofactor in the final step for generating an active enzyme. The gly A gene of Escherichia coli has been shown to code for this enzyme. Analysis of available gene sequences indicate that Serine Hydroxymethyltransferase is one of the most highly conserved proteins. The isolation of the cDNA clones for the enzyme and their overexpression in heterologous systems has enabled the probing of the molecular mechanisms of catalysis and the role of lysine, arginine and histidine in cofactor, substrate(s) binding and in maintaining the structure of the protein. Recently, the three-dimensional structure of the human liver Serine Hydroxymethyltransferase has been published. This, along with the information already available, provides a framework for the rational design of drugs targeted specifically towards this enzyme.
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A change in reaction specificity of sheep liver Serine Hydroxymethyltransferase. Induction of NADH oxidation upon mutation of His230 to Tyr.
European journal of biochemistry, 2000Co-Authors: Rashmi Talwar, N. Appaji Rao, Handanahal S. SavithriAbstract:Both Serine Hydroxymethyltransferase and aspartate aminotransferase belong to the α-class of pyridoxal-5′-phosphate (pyridoxalP)-dependent enzymes but exhibit different reaction and substrate specificities. A comparison of the X-ray structure of these two enzymes reveals that their active sites are nearly superimposable. In an attempt to change the reaction specificity of Serine Hydroxymethyltransferase to a transaminase, His 230 was mutated to Tyr which is the equivalent residue in aspartate aminotransferase. Surprisingly, the H230Y mutant was found to catalyze oxidation of NADH in an enzyme concentration dependent manner instead of utilizing l-aspartate as a substrate. The NADH oxidation could be linked to oxygen consumption or reduction of nitrobluetetrazolium. The reaction was inhibited by radical scavengers like superoxide dismutase and d-mannitol. The Km and kcat values for the reaction of the enzyme with NADH were 74 µm and 5.2 × 10−3 s−1, respectively. This oxidation was not observed with either the wild type Serine Hydroxymethyltransferase or H230A, H230F or H230N mutants. Thus, mutation of H230 of sheep liver Serine Hydroxymethyltransferase to Tyr leads to induction of an NADH oxidation activity implying that tyrosyl radicals may be mediating the reaction.
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The primary structure of sheep liver cytosolic Serine Hydroxymethyltransferase and an analysis of the evolutionary relationships among Serine Hydroxymethyltransferases.
Biochimica et biophysica acta, 1994Co-Authors: Rajagopalan Usha, Handanahal S. Savithri, N. Appaji RaoAbstract:The complete amino-acid sequence of sheep liver cytosolic Serine Hydroxymethyltransferase was determined from an analysis of tryptic, chymotryptic, CNBr and hydroxylamine peptides. Each subunit of sheep liver Serine Hydroxymethyltransferase consisted of 483 amin-acid residues. A comparison of this sequence with 8 other Serine Hydroxymethyltransferases revealed that a possible gene duplication event could have occurred after the divergence of animals and fungi. This analysis also showed independent duplication of SHMT genes in Neurospora crassa. At the secondary structural level, all the Serine Hydroxymethyltransferases belong to the α/β category of proteins. The predicted secondary structure of sheep liver Serine Hydroxymethyltransferase was similar to that of the observed structure of tryptophan synthase, another pyridoxal 5′-phosphate containing enzyme, suggesting that sheep liver Serine Hydroxymethyltransferase might have a similar pyridoxal 5′-phosphate binding domain. In addition, a conserved glycinerich region, G L Q G G P, was identified in all the Serine Hydroxymethyltransferases and could be important in pyridoxal 5′-phosphate binding. A comparison of the cytosolic Serine Hydroxymethyltransferases from rabbit and sheep liver with other proteins sequenced from both these sources showed that Serine Hydroxymethyltransferase was a highly conserved protein. In was slightly less conserved than cytochrome c but better conserved than myoglobin, both of which are well known evolutionary markers. C67 and C203 were specifically protected by pyridoxal 5′-phosphate against modification with [14C]iodoacetic acid, while C247 and C261 were buried in the native Serine Hydroxymethyltransferase. However, the cysteines are not conserved among the various Serine Hydroxymethyltransferases. The exact role of the cysteines in the reaction catalyzed by Serine Hydroxymethyltransferase remains to be elucidated.
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Serine Hydroxymethyltransferase from Mung Bean (Vigna radiata) Is Not a Pyridoxal-5'-Phosphate-Dependent Enzyme.
Plant physiology, 1991Co-Authors: Narasimhan Sukanya, Handanahal S. Savithri, M. Vijaya, A. N. Radhakrishnan, N. Appaji RaoAbstract:Serine Hydroxymethyltransferase from mammalian and bacterial sources is a pyridoxal-5′-phosphate-containing enzyme, but the requirement of pyridoxal-5′-phosphate for the activity of the enzyme from plant sources is not clear. The specific activity of Serine Hydroxymethyltransferase isolated from mung bean (Vigna radiata) seedlings in the presence and absence of pyridoxal-5′-phosphate was comparable at every step of the purification procedure. The mung bean enzyme did not show the characteristic visible absorbance spectrum of a pyridoxal-5′-phosphate protein. Unlike the enzymes from sheep, monkey, and human liver, which were converted to the apoenzyme upon treatment with l-cysteine and dialysis, the mung bean enzyme similarly treated was fully active. Additional evidence in support of the suggestion that pyridoxal-5′-phosphate may not be required for the mung bean enzyme was the observation that pencillamine, a well-known inhibitor of pyridoxal-5′-phosphate enzymes, did not perturb the enzyme spectrum or inhibit the activity of mung bean Serine Hydroxymethyltransferase. The sheep liver enzyme upon interaction with O-amino-d-Serine gave a fluorescence spectrum with an emission maximum at 455 nm when excited at 360 nm. A 100-fold higher concentration of mung bean enzyme-O-amino-d-Serine complex did not yield a fluorescence spectrum. The following observations suggest that pyridoxal-5′-phosphate normally present as a coenzyme in Serine Hydroxymethyltransferase was probably replaced in mung bean Serine Hydroxymethyltransferase by a covalently bound carbonyl group: (a) inhibition by phenylhydrazine and hydroxylamine, which could not be reversed by dialysis and or addition of pyridoxal-5′ phosphate; (b) irreversible inactivation by sodium borohydride; (c) a spectrum characteristic of a phenylhydrazone upon interaction with phenylhydrazine; and (d) the covalent labeling of the enzyme with substrate/product Serine and glycine upon reduction with sodium borohydride. These results indicate that in mung bean Serine Hydroxymethyltransferase, a covalently bound carbonyl group has probably replaced the pyridoxal-5′-phosphate that is present in the mammalian and bacterial enzymes.
Francesco Bossa - One of the best experts on this subject based on the ideXlab platform.
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Structural adaptation of Serine Hydroxymethyltransferase to low temperatures.
International Journal of Biological Macromolecules, 2009Co-Authors: Alessandro Siglioccolo, Francesco Bossa, Stefano PascarellaAbstract:Structural adaptation of Serine Hydroxymethyltransferase (SHMT), a pyridoxal-5′-phosphate dependent enzyme that catalyzes the reversible conversion of l-Serine and tetrahydropteroylglutamate to glycine and 5,10-methylene-tetrahydropteroylglutamate, synthesized by microorganisms adapted to low temperatures has been analyzed using a comparative approach. The variations of amino acid properties and frequencies among three temperature populations (psychrophilic, mesophilic, hyper- and thermophilic) of SHMT sequences have been tested. SHMTs display a general increase of polarity specially in the core, a more negatively charged surface, and enhanced flexibility. Subunit interface is more hydrophilic and less compact. Electrostatic potential of the tetrahydrofolate binding site has been compared. The enzyme from Psychromonas ingrahamii, the organism with the lowest adaptation temperatures, displayed the most positive potential. In general, the property variations show a coherent opposite trend in the hyperthermophilic population: in particular, increase of hydrophobicity, packing and decrease of flexibility was observed.
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Role of tyrosine 65 in the mechanism of Serine Hydroxymethyltransferase.
Biochemistry, 2000Co-Authors: Roberto Contestabile, Sebastiana Angelaccio, Francesco Bossa, G. Kazanina, H.t. Wright, N. Scarsdale, Verne SchirchAbstract: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. ...
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Role of Y65 and E57 in Escherichia coli Serine Hydroxymethyltransferase
Biochemistry and Molecular Biology of Vitamin B6 and PQQ-dependent Proteins, 2000Co-Authors: Sebastiana Angelaccio, Roberto Contestabile, Francesco Bossa, P. Di Giovine, Verne SchirchAbstract:The crystal structures of Serine Hydroxymethyltransferase from three different sources have recently been solved. Inspection of the active site reveals that, besides K229, whose role as catalytic base had been ruled out, either E57 or Y65 may be the putative active-site proton exchanger required for catalysis. Two mutant forms of theE. co/ienzyme, E57Q and Y65F, were produced and their catalytic properties studied. The results show that neither E57 nor Y65 is the elusive catalytic base. The Y65F mutation substantially reduced Keatand Kmfor cleavage of β-hydroxyamino acids, although the catalytic efficiency was unaffected. A possible role of Y65 in the conformational change that is thought to take place upon binding of substrates is proposed.
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Deamidation of Asparagine Residues in a Recombinant Serine Hydroxymethyltransferase
Archives of Biochemistry and Biophysics, 1999Co-Authors: Martino L. Di Salvo, Sonia Delle Fratte, H. Tonie Wright, Francesco Bossa, Bruno Maras, Verne SchirchAbstract:Serine Hydroxymethyltransferase purified from rabbit liver cytosol has at least two Asn residues (Asn5 and Asn220) that are 67 and 30% deamidated, respectively. Asn5 is deamidated equally to Asp and isoAsp, while Asn220 is deamidated only to isoAsp. To determine the effect of these Asn deamidations on enzyme activity and stability a recombinant rabbit liver cytosolic Serine Hydroxymethyltransferase was expressed in Escherichia coli over a 5-h period. About 90% of the recombinant enzyme could be isolated with the two Asn residues in a nondeamidated form. Compared with the enzyme isolated from liver the recombinant enzyme had a 35% increase in catalytic activity but exhibited no significant changes in either affinity for substrates or stability. Introduction of Asp residues for either Asn5 or Asn220 did not significantly alter activity or stability of the mutant forms. In vitro incubation of the recombinant enzyme at 37°C and pH 7.3 resulted in the rapid deamidation of Asn5 to both Asp and isoAsp with a t1/2 of 50–70 h, which is comparable to the rate found with small flexible peptides containing the same sequence. The t1/2 for deamidation of Asn220 was at least 200 h. This residue may become deamidated only after some unfolding of the enzyme. The rates for deamidation of Asn5 and Asn220 are consistent with the structural environment of the two Asn residues in the native enzyme. There are also at least two additional deamidation events that occur during prolonged incubation of the recombinant enzyme.
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Purification and Characterization of Recombinant Rabbit Cytosolic Serine Hydroxymethyltransferase
Protein expression and purification, 1998Co-Authors: Martino L. Di Salvo, Sonia Delle Fratte, Francesco Bossa, Daniela De Biase, Verne SchirchAbstract:Abstract A rabbit liver cDNA library in phage λgt10 was screened using the coding cDNA for human cytosolic Serine Hydroxymethyltransferase. A clone of 1754 bp was isolated and the nucleotide sequence showed an open reading frame of 1455 bp, which coded for rabbit cytosolic Serine Hydroxymethyltransferase and was flanked by 12 bp at the 5′ end and 287 bp at the 3′ end. The full-length cDNA was then cloned into a pET22b vector as a Nde I– Eco RI insert. HMS174(DE3) cells were transformed with this plasmid and, after induction with isopropyl β- d -thiogalactopyranoside, expressed a catalytically active Serine Hydroxymethyltransferase. The enzyme was purified and shown to be the expressed rabbit enzyme lacking the first methionine residue. Spectral characteristics of the bound pyridoxal phosphate and kinetic constants for the natural substrates l -Serine and tetrahydrofolate were essentially identical to the values obtained previously for the rabbit cytosolic enzyme. The pattern of bands shown by the pure recombinant enzyme on an isoelectric focusing gel containing 6 M urea showed a major band and a minor band representing about 15–20% of the protein. Upon incubation of the recombinant enzyme at pH 7.3 and 37°C, three new bands were observed on isoelectric focusing with the concomitant formation of isoaspartyl residues, as determined by reactivity with protein isoaspartyl methyltransferase. These results are consistent with deamidation of Asn residues to isoaspartyl during the in vitro incubation. The enzyme purified from rabbit liver has previously been shown to contain isoaspartyl residues.
