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.     

Identification and localization of multiple forms of serine hydroxymethyltransferase in pea (Pisum sativum) and characterization of a cDNA encoding a mitochondrial isoform.

Serine hydroxymethyltransferase (SHMT) has been purified from the mitochondria of green pea leaves. Activity can be fractionated into two distinct peaks by ion exchange chromatography. While these two forms of the enzyme are immunologically indistinguishable, immunoinhibition experiments show the presence of a distinct non-mitochondrial third form of the enzyme to also be present in green pea leaves. While this mitochondrial form of SHMT is abundant in leaves it is absent from roots, although the two tissues have comparable SHMT activity. An antibody raised to purified mitochondrial SHMT was used to screen a cDNA expression library. The sequence of one of the isolated positive clones contained an open reading frame, which encoded a sequence that matched the amino acid sequence determined from the N terminus of the mature protein. The open reading frame encodes a mature protein of 487 amino acids with a M(r) of 54,000, together with a 27-31 amino acid serine-rich leader sequence, presumably required for mitochondrial targeting. The cDNA hybridizes to a small multigene family of 2-3 genes, which appear to be expressed predominantly in leaves. Comparison of the deduced amino acid sequence with the amino acid sequences of the rabbit mitochondrial and cytoplasmic SHMT, show that pea mitochondrial SHMT is equally similar to both of these enzymes. In addition, the rabbit sequences are more like one another than they are to the pea sequence, suggesting an interesting evolutionary relationship for these proteins.     

Unusual structural,functional, and stability properties of serine hydroxymethyltransferase from Mycobacterium tuberculosis

From the genome analysis of the Mycobacterium tuberculosis two putative genes namely GlyA and GlyA2 have been proposed to encode for the enzyme serine hydroxymethyltransferase. We have cloned, overexpressed, and purified to homogeneity their respective protein products, serine hydroxymethyltransferase, SHM1 and SHM2. The recombinant SHM1 and SHM2 exist as homodimers of molecular mass about 90 kDa under physiological conditions, however, SHM2 has more compact conformation and higher thermal stability than SHM1. The most interesting structural observation was that the SHM1 contains 1 mol of pyridoxal 5'-phosphate (PLP)/mol of enzyme dimer. This is the first report of such a unique stoichiometry of PLP and enzyme dimer for SHMT. The SHM2 contains 2 mol of PLP/mol of enzyme dimer, which is the usual stoichiometry reported for SHMT. Functionally both the recombinant enzymes showed catalysis of reversible interconversion of serine and glycine and aldol cleavage of a 3-hydroxyamino acid. However, unlike SHMT from other sources both SHM1 and SHM2 do not undergo half-transamination reaction with d-alanine resulting in formation of apoenzyme but l-cysteine removed the prosthetic group, PLP, from both the recombinant enzymes leaving the respective inactive apoenzymes. Comparative structural studies on the two enzymes showed that the SHM1 is resistant to alkaline denaturation up to pH 10.5, whereas the native SHM2 dimer dissociates into monomer at pH 9. Urea- and guanidinium chloride-induced two-step unfolding of SHM1 and SHM2 with the first step being dissociation of dimer into apomonomer at low denaturant concentrations followed by unfolding of the stabilized monomer at higher denaturant concentrations.     

DNA enzymes

The past year has seen a coming-of-age in DNA enzyme research. Far from being laboratory curiosities, the activities of new DNA enzymes have broadened the known catalytic repertoire of nucleic acid enzymes, provided valuable insights into different mechanistic possiblities open to nucleic acid catalysts, and explored the importance for catalysis of native functionalities within DNA and RNA, as well as of a diversity of extrinsic cofactors. Thus, the first amino acid cofactor-utilizing DNA enzyme has been described, as well as DNA enzymes that cleave RNA without the assistance of any external cofactor. On the practical side, the most efficient RNA-cleaving nucleic acid enzyme described to date is a DNA enzyme.     

Identification and biochemical characterization of serine hydroxymethyl transferase in the hydrogenosome of Trichomonas vaginalis

Serine hydroxymethyl transferase (SHMT) is a pyridoxal phosphate (PLP)-dependent enzyme that catalyzes the reversible conversion of serine and tetrahydrofolate to glycine and methylenetetrahydrofolate. We have identified a single gene encoding SHMT in the genome of Trichomonas vaginalis, an amitochondriate, deep-branching unicellular protist. The protein possesses a putative N-terminal hydrogenosomal presequence and was shown to localize to hydrogensomes by immunofluorescence analysis, providing evidence of amino acid metabolism in this unusual organelle. In contrast to the tetrameric SHMT that exists in the mammalian host, we found that the T. vaginalis SHMT is a homodimer, as found in prokaryotes. All examined SHMT contain an 8-amino-acid conserved sequence, VTTTTHKT, containing the active-site lysyl residue (Lys 251 in TvSHMT) that forms an internal aldimine with PLP. We mutated this Lys residue to Arg and Gln and examined structural and catalytic properties of the wild-type and mutant enzymes in comparison to that reported for the mammalian protein. The oligomeric structure of the mutant K251R and K251Q TvSHMT was not affected, in contrast to that observed for comparable mutations in the mammalian enzyme. Likewise, contrary to that observed for mammalian SHMT, the catalytic activity of K251R TvSHMT was unaffected in the presence of PLP. The K251Q TvSHMT, however, was found to be inactive. These studies indicate that the active site of the parasite enzyme is distinct from its prokaryotic and eukaryotic counterparts and identify TvSHMT as a potential drug target.     

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.     

Evolution of the biosynthesis of the branched-chain amino acids

The origin of the biosynthetic pathways for the branched-chain amino acids cannot be understood in terms of the backwards development of the present acetolactate pathway because it contains unstable intermediates. We propose that the first biosynthesis of the branched-chain amino acids was by the reductive carboxylation of short branched chain fatty acids giving keto acids which were then transaminated. Similar reaction sequences mediated by nonspecific enzymes would produce serine and threonine from the abundant prebiotic compounds glycolic and lactic acids. The aromatic amino acids may also have first been synthesized in this way, e.g. tryptophan from indole acetic acid. The next step would have been the biosynthesis of leucine from -ketoisovaleric acid. The acetolactate pathway developed subsequently. The first version of the Krebs cycle, which was used for amino acid biosynthesis, would have been assembled by making use of the reductive carboxylation and leucine biosynthesis enzymes, and completed with the development of a single new enzyme, succinate dehydrogenase. This evolutionary scheme suggests that there may be limitations to inferring the origins of metabolism by a simple back extrapolation of current pathways.     

Cloning and characterization of Plasmodium vivax serine hydroxymethyltransferase

Serine hydroxymethyltransferase (SHMT), which catalyzes the reversible reaction of serine and tetrahydrofolate to glycine and methylenetetrahydrofolate, is one of the three enzymes in dTMP synthesis pathway that is highly active during cell division and has been proposed as a potential chemotherapeutic target in infectious diseases and cancer. This is the first study to describe nucleotide and amino acid sequences of SHMT from the malaria parasite Plasmodium vivax. Sequencing of 12 P. vivax isolates revealed limited polymorphisms in 3 noncoding regions. Its biological function is also reported.     

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.     

l-Serine production by a methylotroph and its related enzymes

The production process of l-serine from methanol and glycine has been developed using a methylotroph with the serine pathway. Consecutive reactions of two enzymes, methanol dehydrogenase (MDH) and serine hydroxymethyltransferase (SHMT) are involved in the production. We screened a high producer, Hyphomicrobium methylovorum, which is an obligate methylotroph. With resting cells of the bacterium, 24 mg/ml of l-serine was produced from 100 mg/ml of glycine and 48 mg/ml of methanol in 3 days under optimal conditions. Next, a glycine-resistant mutant GM2 showed improved serine production (32–34 mg/ml). The mutant GM2 was found to have elevated activities of MDH and SHMT. Since there has so far been little report on the systematic characterization of enzymes of the serine pathway in methylotrophs, not only the above two enzymes but also the other three enzymes in H. methylovorum were purified and characterized: MDH, SHMT and hydroxypyruvate reductase (HPR) were crystallized; serine-glyoxylate aminotransferase (SGAT) and glycerate kinase (GK) were purified to homogeneity. As a result, all these enzymes were found to be stable against preservation and to exist abundantly in the bacterium. The gene of SHMT was cloned and its deduced amino acid sequence had homology to those of Escherichia coli (55%) and rabbit liver (44%), whereas the enzyme of the bacterium was immunochemically distinguishable from those of microorganisms other than Hyphomicrobium strains and mammalian livers. Correspondence to: Y. Izumi     

Isolation and sequence analysis of a complementary DNA encoding rat liver L-gulono-gamma-lactone oxidase, a key enzyme for L-ascorbic acid biosynthesis

L-Gulono-gamma-lactone oxidase, one of the microsomal flavin enzymes, catalyzes the last step of L-ascorbic acid biosynthesis in many animals; however, it is missing in scurvy-prone animals such as humans, primates, and guinea pigs. A cDNA clone for this enzyme was isolated by screening a rat liver cDNA expression library in lambda gt11 using antibody directed against the enzyme. The cDNA clone contained 2120 nucleotides and an open reading frame of 1320 nucleotides encoding 440 amino acids of the protein with a molecular weight of 50,605. The amino-terminal sequence (residues 1-33) of the enzyme isolated from rat liver completely coincided with the corresponding part of the deduced amino acid sequence. The identity of the cDNA clone was further confirmed by the agreement of the composition of the deduced amino acids with that determined by amino acid analysis of the enzyme. Hydropathy analysis of the deduced amino acid sequence revealed several hydrophobic regions, suggesting that they anchor the protein into the microsomal membrane. The deduced amino acid sequence showed no obvious homology with the flavin-binding regions of other eight flavoenzymes.     

Expression,purification, and characterization of bovine chymosin enzyme using an inducible pTOLT system

In recent years, various studies in the field of industrial enzymes of biotechnology have gained importance due to increasing development in enzyme technology. The different areas where enzymes are used and their economic value of biotechnological products further increases their importance. There are hundreds of different types of cheese but each is made by coagulating milk using rennet to give curds. Today, researchers have begun to develop alternative systems in the cheese industry related to milk-clotting enzymes. In this study, the nucleic acid sequence encoding the optimized chymosin enzyme was used and cloned by Not I and Mlu I restriction enzymes into pTOLT vector system. Then using this construct, the enzyme as a fusion with Tol-A-III protein was produced in Escherichia coli BL21 (DE3) cells. After disrupting the E. coli cell and separating from the constituents by high speed centrifugation, the enzyme was purified by affinity chromatography and fractions were analyzed by SDS–PAGE. Purified enzyme has shown its activity. Optimum temperature and pH of CHY-Tol-A-III protein were 40°C and 6.5, respectively.     

The RAGNYA fold: a novel fold with multiple topological variants found in functionally diverse nucleic acid, nucleotide and peptide-binding proteins

Using sensitive structure similarity searches, we identify a shared alpha+beta fold, RAGNYA, principally involved in nucleic acid, nucleotide or peptide interactions in a diverse group of proteins. These include the Ribosomal proteins L3 and L1, ATP-grasp modules, the GYF domain, DNA-recombination proteins of the NinB family from caudate bacteriophages, the C-terminal DNA-interacting domain of the Y-family DNA polymerases, the uncharacterized enzyme AMMECR1, the siRNA silencing repressor of tombusviruses, tRNA Wybutosine biosynthesis enzyme Tyw3p, DNA/RNA ligases and related nucleotidyltransferases and the Enhancer of rudimentary proteins. This fold exhibits three distinct circularly permuted versions and is composed of an internal repeat of a unit with two-strands and a helix. We show that despite considerable structural diversity in the fold, its representatives show a common mode of nucleic acid or nucleotide interaction via the exposed face of the sheet. Using this information and sensitive profile-based sequence searches: (1) we predict the active site, and mode of substrate interaction of the Wybutosine biosynthesis enzyme, Tyw3p, and a potential catalytic role for AMMECR1. (2) We provide insights regarding the mode of nucleic acid interaction of the NinB proteins, and the evolution of the active site of classical ATP-grasp enzymes and DNA/RNA ligases. (3) We also present evidence for a bacterial origin of the GYF domain and propose how this version of the fold might have been utilized in peptide interactions in the context of nucleoprotein complexes.     

Non-ribosomal biosynthesis of the cyclic octadecapeptide alamethicin.

The biosynthesis of the cyclic octadecapeptide, alamethicin, in a cell-free system of Trichoderma viride has been investigated. It was shown that nucleic acid- and ribo-some-free extracts of Trichoderma viride could catalyze alamethicin biosynthesis. Puromycin, erythromycin and RNAse did not inhibit this synthesis. The Sephadex G 200 filtrate contains a fraction (Kav=0.1) that catalyzes the biosynthesis of alamethicin and shows an ATP-32PPi exchange with 6 of the 8 constituent amino acids of alamethicin. The activated amino acids are bound to the enzyme as aminoacyl adenylates and as thiolesters in a proportion of 1 : 1. About 50% of each bound amino acid could be split off with 7% TCA. The TCA-stable bound amino acid could be split by mercury acetate, hydroxylamine and performic acid. N-ethylmaleimide blocked the binding of 50% of the amino acids to the enzyme, proving that some of the amino acids first bound as aminoacyl adenylates are then transferred into a thiolester bond.     

The Saccharomyces cerevisiae ARO1 gene. An example of the co-ordinate regulation of five enzymes on a single biosynthetic pathway

The ARO1 gene of Saccharomyces cerevisiae encodes the arom multifunctional enzyme. Specific inhibitors of amino acid biosynthesis have been used to obtain evidence that expression of a cloned ARO1 gene is regulated in response to amino acid limitation. Northern blot analysis and sequence studies indicate that ARO1 is regulated by the well characterised S. cerevisiae 'general control' mechanism. This provides a very economical means of simultaneously tailoring the synthesis of five shikimate pathway enzymes to the needs of the cell.     

Serine hydroxymethyltransferase anchors de novo thymidylate synthesis pathway to nuclear lamina for DNA synthesis

The de novo thymidylate biosynthetic pathway in mammalian cells translocates to the nucleus for DNA replication and repair and consists of the enzymes serine hydroxymethyltransferase 1 and 2α (SHMT1 and SHMT2α), thymidylate synthase, and dihydrofolate reductase. In this study, we demonstrate that this pathway forms a multienzyme complex that is associated with the nuclear lamina. SHMT1 or SHMT2α is required for co-localization of dihydrofolate reductase, SHMT, and thymidylate synthase to the nuclear lamina, indicating that SHMT serves as scaffold protein that is essential for complex formation. The metabolic complex is enriched at sites of DNA replication initiation and associated with proliferating cell nuclear antigen and other components of the DNA replication machinery. These data provide a mechanism for previous studies demonstrating that SHMT expression is rate-limiting for de novo thymidylate synthesis and indicate that de novo thymidylate biosynthesis occurs at replication forks.     

Regulation of enzyme synthesis in the aromatic amino acid pathway of Bacillus subtilus

The control of the synthesis of certain key enzymes of aromatic amino acid biosynthesis was studied. Tyrosine represses the first enzyme of the 3-deoxy-d-arabino heptulosonic acid 7-phosphate pathway, DAHP synthetase, as well as shikimate kinase and chorismate mutase about fivefold in cultures grown under conditions limiting the synthesis of the aromatic amino acids. A mixture of tyrosine and phenylalanine represses twofold further. Tryptophan does not appear to be involved in the control of these enzymes. The specific activity of at least one early enzyme, dehydroquinase, remains essentially constant under a variety of nutritional supplementations. Two enzymes in the terminal branches are repressed by the amino acids they help to synthesize: prephenate dehydrogenase can be repressed fourfold by tyrosine, and anthranilate synthetase can be repressed over 200-fold by tryptophan. There is no evidence that phenylalanine represses prephenate dehydratase. Regulatory mutants have been isolated in which various enzymes of the pathway are no longer repressible. One class is derepressed for several of the prechorismate enzymes, as well as chorismate mutase and prephenate dehydrogenase. In another mutant, several enzymes of tryptophan biosynthesis are no longer repressible. Thus, the rate of synthesis of enzymes at every stage of the pathway is under control of various aromatic amino acids. Tyrosine and phenylalanine control the synthesis of enzymes involved in the synthesis of the three aromatic amino acids. Each terminal branch is under the control of its end product.     

Biosynthesis of lysosomal endopeptidases

Despite the clear differences between the amino acid sequence and enzymatic specificity of aspartic and cysteine endopeptidases, the biosynthetic processing of lysosomal members of these two families is very similar. With in vitro translation and pulse-chase analysis in tissue culture cells, the biosynthesis of cathepsin D, a aspartic protease, and cathepsins B, H and L, cysteine proteases, are compared. Both aspartic and cysteine endopeptidases undergo cotranslational cleavage of an amino-terminal signal peptide that mediates transport across the endoplasmic reticulum (ER) membrane. Addition of high-mannose carbohydrate also occurs cotranslationally in the lumen of the ER. Proteases of both enzyme classes are initially synthesized as inactive proenzymes possessing amino-terminal activation peptides. Removal of the propeptide generates an active single-chain enzyme. Whether the single-chain enzyme undergoes asymmetric cleavage into a light and a heavy chain appears to be cell type specific. Finally, late during their biosynthesis both classes of enzymes undergo amino acid trimming, losing a few amino acid residues at the cleavage site between the light and heavy chains and/or at their carboxyltermini. During biosynthesis these enzymes are also secreted to some extent. In most cells the secreted enzyme is the proenzyme bearing some complex carbohydrate. Under certain physiological conditions the inactive secreted enzymes may become activated as a result of a conformational change that may or may not result in autolysis. Analysis of the biochemical nature of the various processing steps helps define the cellular pathway followed by newly synthesized proteases targeted to the lysosome.     

The Crystal Structure of the Adenylation Enzyme VinN Reveals a Unique β-Amino Acid Recognition Mechanism

Adenylation enzymes play important roles in the biosynthesis and degradation of primary and secondary metabolites. Mechanistic insights into the recognition of α-amino acid substrates have been obtained for α-amino acid adenylation enzymes. The Asp residue is invariant and is essential for the stabilization of the α-amino group of the substrate. In contrast, the β-amino acid recognition mechanism of adenylation enzymes is still unclear despite the importance of β-amino acid activation for the biosynthesis of various natural products. Herein, we report the crystal structure of the stand-alone adenylation enzyme VinN, which specifically activates (2S,3S)-3-methylaspartate (3-MeAsp) in vicenistatin biosynthesis. VinN has an overall structure similar to that of other adenylation enzymes. The structure of the complex with 3-MeAsp revealed that a conserved Asp²³⁰ residue is used in the recognition of the β-amino group of 3-MeAsp similar to α-amino acid adenylation enzymes. A mutational analysis and structural comparison with α-amino acid adenylation enzymes showed that the substrate-binding pocket of VinN has a unique architecture to accommodate 3-MeAsp as a β-amino acid substrate. Thus, the VinN structure allows the first visualization of the interaction of an adenylation enzyme with a β-amino acid and provides new mechanistic insights into the selective recognition of β-amino acids in this family of enzymes.     

A functional screen for Myc-responsive genes reveals serine hydroxymethyltransferase,a major source of the one-carbon unit for cell metabolism

A cDNA library enriched with Myc-responsive cDNAs but depleted of myc cDNAs was used in a functional screen for growth enhancement in c-myc-null cells. A cDNA clone for mitochondrial serine hydroxymethyltransferase (mSHMT) that was capable of partial complementation of the growth defects of c-myc-null cells was identified. Expression analysis and chromatin immunoprecipitation demonstrated that mSHMT is a direct Myc target gene. Furthermore, a separate gene encoding the cytoplasmic isoform of the same enzyme is also a direct target of Myc regulation. SHMT enzymes are the major source of the one-carbon unit required for folate metabolism and for the biosynthesis of nucleotides and amino acids. Our data establish a novel functional link between Myc and the regulation of cellular metabolism.