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Query: UNIPROT:P06889 (Mol)
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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.
J Mol Biol 2000 Feb 11
PMID:Crystal structure at 2.4 A resolution of E. coli serine hydroxymethyltransferase in complex with glycine substrate and 5-formyl tetrahydrofolate. 1065 24

The Fourier transform infrared and Raman spectra of di-i-propoxyphosphoryl benzylisothiourea (DPB) (1) in the solid state and in solutions of CCl4, CHCl3, CHBr3, CH2Cl2, C2H4Cl2, C2H4Br2 and THF were studied. In the IR spectra, the effects of different concentrations were also investigated. The behavior of the nu(NH), delta(NH), delta(HNH), nu(C=N) and nu(P=O) normal modes suggests the existence of a tautomerism between the phosphorylamine (I) and N-phosphorylimine (II) structures: [structures: see text] The data show the presence of different delta(NH) and delta(HNH) bendings and nu(C=N) normal modes in the solid state as a result of inter and intramolecular hydrogen bonding. The experimental approximate frequencies assignments were done for this compound, and were confirmed by a normal coordinate analysis carried out for several fragments of phosphorylamine and N-phosphorylimine structures.
Spectrochim Acta A Mol Biomol Spectrosc 2000 Mar
PMID:A vibrational study of di-i-propoxyphosphoryl benzylisothiourea. 1079 42

Comparison of the three-dimensional structures of folylpolyglutamate synthetase (FPGS) and the bacterial cell wall ligase UDP-N-acetylmuramoyl-l-alanine:d-glutamate ligase (MurD) reveals that these two enzymes have a remarkable structural similarity despite a low level of sequence identity. Both enzymes have a modular, multi-domain structure and catalyse a similar ATP-dependent reaction involving the addition of a glutamate residue to a carboxylate-containing substrate, tetrahydrofolate in the case of FPGS, and UDP-N-acetylmuramoyl-l-alanine in the case of MurD. Site-directed mutations of selected residues in the active site of Lactobacillus casei FPGS (P74A, E143A, E143D, E143Q, K185A, D313A, H316A, G411A and S412A) showed that most of these changes resulted in an almost complete loss of activity. Several of these amino acid residues in FPGS are found in structurally equivalent positions to active-site residues in MurD. Some insights into the function of these residues in FPGS activity are proposed, based on the roles surmised from the structures of two MurD. UDP-N-acetylmuramoyl-l-alanine.ADP complexes and a MurD. UDP-N-acetylmuramoyl-l-alanine-d-glutamate complex. Furthermore, the comparison has led us to propose that conformational changes induced by substrate binding in the reaction mechanism of FPGS result in a movement of the domains towards each other to more closely resemble the orientation of the corresponding domains in MurD. This relative domain movement may be a key feature of this new family of ADP-forming amide bond ligases.
J Mol Biol 2000 Sep 15
PMID:Structural and functional similarities in the ADP-forming amide bond ligase superfamily: implications for a substrate-induced conformational change in folylpolyglutamate synthetase. 1097 Jul 43

The photophysical processes of copolymer formed by copolymerization of beta-naphthyl-methacrylate (NMA) with vinylcarbazole (VCZ) were studied. The results show that when the solution of copolymer (NMA-VCZ) in THF is located in a low concentration range (about 10(-8) mol/l), the fluorescence emission is in good agreement with that of NMA monomer and the excimer is formed with gradual increase in concentration of copolymer (NMA-VCZ). The fluorescence of copolymer (NMA-VCZ) can be efficiently quenched both by electron donors and acceptors where the quenching effects follow the Stern-Volmer equation. The dimolecular exciplex between copolymer (NMA-VCZ) and N,N-dimethylaniline (DMA) is formed and the triple exciplex is also observed in the same system.
Spectrochim Acta A Mol Biomol Spectrosc 2000 Sep
PMID:Photophysical processes of a copolymer containing naphthalene and carbazole rings. 1098 78

Alterations in homocysteine, methionine, folate, and/or B12 homeostasis have been associated with neural tube defects, cardiovascular disease, and cancer. Methionine synthase, one of only two mammalian enzymes known to require vitamin B12 as a cofactor, lies at the intersection of these metabolic pathways. This enzyme catalyzes the transfer of a methyl group from 5-methyl-tetrahydrofolate to homocysteine, generating tetrahydrofolate and methionine. Human patients with methionine synthase deficiency exhibit homocysteinemia, homocysteinuria, and hypomethioninemia. They suffer from megaloblastic anemia with or without some degree of neural dysfunction and mental retardation. To better study the pathophysiology of methionine synthase deficiency, we utilized gene-targeting technology to inactivate the methionine synthase gene in mice. On average, heterozygous knockout mice from an outbred background have slightly elevated plasma homocysteine and methionine compared to wild-type mice but seem to be otherwise indistinguishable. Homozygous knockout embryos survive through implantation but die soon thereafter. Nutritional supplementation during pregnancy was unable to rescue embryos that were completely deficient in methionine synthase. Whether any human patients with methionine synthase deficiency have a complete absence of enzyme activity is unclear. These results demonstrate the importance of this enzyme for early development in mice and suggest either that methionine synthase-deficient patients have residual methionine synthase activity or that humans have a compensatory mechanism that is absent in mice.
Mol Cell Biol 2001 Feb
PMID:Targeted disruption of the methionine synthase gene in mice. 1115 93

Neural tube defects (NTD) arise in the first weeks of pregnancy due to a combination of environmental and genetic factors. In mothers of children with NTD elevated homocysteine (Hcy) levels and decreased plasma folate levels were observed, which suggests a defect in the folate-dependent Hcy metabolism. Therefore, mutations in genes coding for enzymes of this metabolism could be involved in NTD. Serine hydroxymethyltransferase (SHMT) catalyzes the reversible reaction of serine and tetrahydrofolate (THF) to glycine and 5,10-methylene THF. Two different isoforms of SHMT are known, one is present in the cytosol (cSHMT) and the other in the mitochondrion (mSHMT). Theoretically, mutated SHMT could lead to elevated Hcy levels and to an altered distribution of the different folate derivatives and might therefore become a risk factor for NTD. This study concerns the molecular genetic analysis of genes coding for both isoforms of the SHMT enzyme by single-stranded conformation polymorphism analysis. Several mutations as well as polymorphisms were found in both genes. The relevance of two variations, the 1420 C>T mutation of the cytosolic isoform and the 4-bp deletion of the mitochondrial isoform (delTCTT 1721-1724), to NTD risk was tested in a study group, which consisted of 109 NTD patients, 120 mothers of children with NTD, and 420 controls. Neither of the two polymorphisms led to an increased risk of NTD. In mothers with the 1420 CC genotype, significant increased Hcy levels are present. Also, significantly decreased red blood cell folate and plasma folate levels were present in individuals with the 1420 CC genotype. Probably, the 1420 C>T polymorphism causes a shift in distribution of the different folate derivatives. The 4-bp deletion of the mSHMT gene did not lead to altered Hcy or folate levels. So far, the results of this study provide no direct evidence for a role of defective SHMT functioning in NTD. Still, the influence of the 1420 C>T polymorphism of the cSHMT gene on the folate-related risk of NTD needs further investigation.
Mol Genet Metab 2001 Jun
PMID:Is mutated serine hydroxymethyltransferase (SHMT) involved in the etiology of neural tube defects? 1138 52

The natural folates are water-soluble members of the B class of vitamins that are essential for cell proliferation and tissue regeneration. Since mammalian cells cannot synthesize folates de novo, tightly regulated and sophisticated cellular uptake processes have evolved to sustain sufficient levels of intracellular tetrahydrofolate cofactors to support the biosynthesis of purines, pyrimidines, serine, and methione. Membrane transport is also a critical determinant of the antitumor activity of antifolate therapeutics (methotrexate, Tomudex) used in cancer chemotherapy, and impaired uptake of antifolates is a frequent mode of drug resistance. The reduced folate carrier is the major transport system for folates and classical antifolates in mammalian cells and tissues. This review summarizes the remarkable advances in the cellular and molecular biology of the human reduced folate carrier over the past decade, relating to its molecular structure and transport function, mechanisms of transcriptional and posttranscriptional regulation, and its critical role in antifolate response and resistance. Many key in vitro findings have now begun to be extended to studies of reduced folate carrier levels and function in patient specimens, paving the way for translating basic laboratory studies in cultured cells to improvements in human health and treatment of disease. The results of research into the human reduced folate carrier should clarify the roles of changes in expression and function of this system that accompany nutritional folate deficiency and human disease, and may lead to improved therapeutic strategies for enhancing drug response and circumventing resistance in cancer patients undergoing chemotherapy with antifolates.
Prog Nucleic Acid Res Mol Biol 2001
PMID:Molecular and cellular biology of the human reduced folate carrier. 1152 81

GTP cyclohydrolase I catalyses the transformation of GTP into dihydroneopterin 3'-triphosphate, which is the first committed precursor of tetrahydrofolate and tetrahydrobiopterin. The kinetically competent reaction intermediate, 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone, was used as substrate for single turnover experiments monitored by multiwavelength photometry. The early reaction phase is characterized by the rapid appearance of an optical transient with an absorption maximum centred at 320. This species is likely to represent a Schiff base intermediate at the initial stage of the Amadori rearrangement of the carbohydrate side-chain. Deconvolution of the optical spectra suggested four linearly independent processes. A fifth reaction step was attributed to photodecomposition of the enzyme product. Pre-steady state experiments were also performed with the H179A mutant which can catalyse a reversible conversion of GTP to 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone but is unable to form the final product, dihydroneopterin triphosphate. Optical spectroscopy failed to detect any intermediate in the reversible reaction sequence catalysed by the mutant protein. The data obtained with the wild-type and mutant protein in conjunction with earlier quenched flow studies show that the enzyme-catalysed opening of the imidazole ring of GTP and the hydrolytic release of formate from the resulting formamide type intermediate are both rapid reactions by comparison with the subsequent rearrangement of the carbohydrate side-chain which precedes the formation of the dihydropyrazine ring of dihydroneopterin triphosphate.
J Mol Biol 2002 Feb 22
PMID:Reaction mechanism of GTP cyclohydrolase I: single turnover experiments using a kinetically competent reaction intermediate. 1186 35

Endospore formation in Bacillus subtilis is primarily dependent on the phosphorylation of the key transcription factor Spo0A by two major kinases, KinA and KinB, thought to be activated by distinct signals. Using a strategy designed to detect mutations that specifically affect the signalling pathway to KinB, we have isolated a Tn10 insertion mutant in one of two adjacent lrp-like genes coding for homologues of the Escherichia coli leucine-responsive regulatory protein (Lrp) and another mutant in the glyA gene encoding the serine hydroxymethyl transferase (SHMT). SHMT catalyses interconversion of serine and glycine while transferring the resulting one-carbon unit into the C1 pool through methylene tetrahydrofolate. Sporulation experiments performed in a series of supplemented media indicated that the role of SHMT in the KinB pathway is to feed the pool of C1 units recruited for the biosynthesis of key metabolites, which include the methyl donor S-adenosyl-methionine (SAM). The results of experiments using L-ethionine suggest that SAM is involved in post-synthetic methylation reactions or biosynthesis of metabolites that serve to activate KinB. Truncated LrpA and LrpB peptides that have retained the DNA-binding domain but have lost the C-terminal half of the protein appear to act as repressors of glyA transcription and KinB-dependent sporulation. However, deletions of lrpA, lrpB or lrpAB have little effect on glyA transcription or sporulation through KinB, suggesting that other effectors, such as additional Lrp homologues, may act in conjunction with LrpA and LrpB. Our results indicate that lrpA-lrpB together with the biosynthetic glyA gene lie on a common signalling pathway meant to activate the KinB sensor kinase.
Mol Microbiol 1997 Jul
PMID:Alterations in the flow of one-carbon units affect KinB-dependent sporulation in Bacillus subtilis. 1190 25

The Archaeon Methanosarcina mazei and related species are of great ecological importance as they are the only organisms fermenting acetate, methylamines and methanol to methane, carbon dioxide and ammonia (in case of methylamines). Since acetate is the precursor of 60% of the methane produced on earth these organisms contribute significantly to the production of this greenhouse gas, e.g. in rice paddies. The 4,096,345 base pairs circular chromosome of M. mazei is more than twice as large as the genomes of the methanogenic Archaea currently completely sequenced (Bult et al., 1996; Smith et al., 1997). 3,371 open reading frames (ORFs) were identified. Based on currently available sequence data 376 of these ORFs are Methanosarcina-specific and 1,043 ORFs find their closest homologue in the bacterial domain. 544 of these ORFs reach significant similarity values only in the bacterial domain. They include 56 of the 102 transposases, and proteins involved in gluconeogenesis, proline biosynthesis, transport processes, DNA-repair, environmental sensing, gene regulation, and stress response. Striking examples are the occurrence of the bacterial GroEL/GroES chaperone system and the presence of tetrahydrofolate-dependent enzymes. These findings might indicate that lateral gene transfer has played an important evolutionary role in forging the physiology of this metabolically versatile methanogen.
J Mol Microbiol Biotechnol 2002 Jul
PMID:The genome of Methanosarcina mazei: evidence for lateral gene transfer between bacteria and archaea. 1212 24


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