Cloning, expression, activity and folding studies of serine hydroxymethyltransferase: a target enzyme for cancer chemotherapy

All the members of pyridoxal-5'-phosphate-dependent enzymes are involved in the metabolism of amino acids. The sequence homology studies further divide this family into three distinct groups. A fine scrutiny of the reactions catalyzed by these enzymes shows their regio specificity; they have been considered as the largest group of enzymes having tendency to affect the valency of the same carbon atom that carries the amino group forming an amine linkage with the coenzyme. Thus, this group was named 'alpha-class of enzymes'. Serine hydroxymethyltransferase (SHMT) is a member of this alpha-class; it reversibly catalyses the conversion of serine into glycine while the hydroxymethyl group is transferred to 5,6,7,8-tetrahydrofolate. The resultant compound is the sole precursor of purine biosynthesis. Henceforth, this enzyme greatly affects nucleic acid biosynthesis in all the organisms. It is obvious that SHMT plays an indispensable role in nucleic acid biosynthesis; therefore, designing and developing a repressor/inhibitor of the SHMT gene/protein may resolve the problem of drug resistance to cancer chemotherapy. SHMT has been widely studied in many living systems (e.g. Escherichia coli, humans, sheep, rabbits, Trypanosoma, Arabidopsis, peas, tobacco) in terms of its structure, cloning, expression, purification and folding patterns. Such studies have enabled one to assess the pattern of overall kinetic and activity behaviour of the enzyme, which may further help in developing a suitable cancer therapeutic molecule.     

Serine hydroxymethyltransferase: origin of substrate specificity.

All forms of serine hydroxymethyltransferase, for which a primary structure is known, have five threonine residues near the active-site lysyl residue (K229) that forms the internal aldimine with pyridoxal phosphate. For Escherichia coli serine hydroxymethyltransferase each of these threonine residues has been changed to an alanine residue. The resulting five mutant enzymes were purified and characterized with respect to kinetic and spectral properties. The mutant enzymes T224A and T227A showed no significant changes in kinetic and spectral properties compared to the wild-type enzyme. The T225A and T230A enzymes exhibited differences in Km and kcat values but exhibited the same spectral properties as the wild-type enzyme. The four threonine residues at positions 224, 225, 227, and 230 do not play a critical role in the mechanism of the enzyme. The T226A enzyme had nearly normal affinity for substrates and coenzymes but had only 3% of the catalytic activity of the wild-type enzyme. The spectrum of the T226A enzyme in the presence of amino acid substrates showed a large absorption maximum at 343 nm with only a small absorption band at 425 nm, unlike the wild-type enzyme whose enzyme-substrate complexes absorb at 425 nm. Rapid reaction studies showed that when amino acid substrates and substrate analogues were added to the T226A enzyme, the internal aldimine absorbing at 422 nm was rapidly converted to a complex absorbing at 343 nm in a second-order process. This was followed by a very slow first-order formation of a complex absorbing at 425 nm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serine hydroxymethyltransferase from Escherichia coli: purification and properties.

Serine hydroxymethyltransferase from Escherichia coli was purified to homogeneity. The enzyme was a homodimer of identical subunits with a molecular weight of 95,000. The amino acid sequence of the amino and carboxy-terminal ends and the amino acid composition of cysteine-containing tryptic peptides were in agreement with the primary structure proposed for this enzyme from the structure of the glyA gene (M. Plamann, L. Stauffer, M. Urbanowski, and G. Stauffer, Nucleic Acids Res. 11:2065-2074, 1983). The enzyme contained no disulfide bonds but had one sulfhydryl group on the surface of the protein. Several sulfhydryl reagents reacted with this exposed group and inactivated the enzyme. Spectra of the enzyme in the presence of substrates and substrate analogs showed that the enzyme formed the same complexes and in similar relative concentrations as previously observed with the cytosolic and mitochondrial rabbit liver isoenzymes. Kinetic studies with substrates showed that the affinity and synergistic binding of the amino acid and folate substrates were similar to those obtained with the rabbit liver isoenzymes. The enzyme catalyzed the cleavage of threonine, allothreonine, and 3-phenylserine to glycine and the corresponding aldehyde in the absence of tetrahydrofolate. The enzyme was also inactivated by D-alanine caused by the transamination of the active site pyridoxal phosphate to pyridoxamine phosphate. This substrate specificity was also observed with the rabbit liver isoenzymes. We conclude that the reaction mechanism and the active site structure of E. coli serine hydroxymethyltransferase are very similar to the mechanism and structure of the rabbit liver isoenzymes.     

Serine hydroxymethyltransferase from the silkworm Bombyx mori: Identification,distribution, and biochemical characterization

Serine hydroxymethyltransferase (SHMT) catalyzes the interconversion of serine and tetrahydrofolate (THF) to glycine and methylenetetrahydrofolate. cDNA encoding Bombyx mori SHMT (bmSHMT) was cloned and sequenced. The deduced amino acid sequence consisted of 465 amino acids and was found to share homology with other SHMTs. Recombinant bmSHMT was overexpressed in Escherichia coli and purified to homogeneity. The enzyme showed optimum activity at pH 3.0 and 30°C and was stable under acidic conditions. The K_m and k_cat/K_m values for THF in the presence of Nicotinamide adenine dinucleotide phosphate (NADP⁺) were 0.055 mM and 0.081 mM^?1 s^?1, respectively, whereas those toward NADP⁺ were 0.16 mM and 0.018 mM^?1 s^?1 and toward l ‐serine were 1.8 mM and 0.0022 mM^?1 s^?1, respectively. Mutagenesis experiments revealed that His119, His132, and His135 are important for enzymatic activity. Our results provide insight into the roles and regulation mechanism of one‐carbon metabolism in the silkworm B. mori.     

Serine hydroxymethyltransferase from soybean root nodules : purification and kinetic properties

Serine hydroxymethyltransferase has been purified 1,550-fold from the plant fraction of soybean (Glycine max [L]. Merr. cv Williams) nodules. The pH optimum for the enzyme was at 8.5. The native molecular weight was 230,000 with a subunit molecular weight of 55,000 which suggested a tetramer of identical subunits. The enzyme kinetics for the enzyme were Michaelis-Menten; there was no evidence for cooperativity in the binding of either substrates or product inhibitors. There were two K_m values for serine at 1.5 and 40 millimolar. The K_m for l-tetrahydrofolate was 0.25 millimolar. l-Methyl-, l-methenyl-, and l-methylene-tetrahydrofolates were all noncompetitive inhibitors with l-tetrahydrofolate with K_i values of 1.8, 3.0, and 2.9 millimolar, respectively. Glycine was a competitive inhibitor with serine with a K_i value of 3.0 millimolar. The intersecting nature of the double reciprocal plots together with the product inhibition data suggested an ordered mechanism with serine the first substrate to bind and glycine the last product released. The enzyme was insensitive to a wide range of metabolites which have previously been reported to affect its activity. These results are discussed with respect to the roles of serine hydroxymethyltransferase and the one-carbon metabolite pool in control of the carbon flow to the purine biosynthetic pathway in ureide biogenesis.     

Plasmodium serine hydroxymethyltransferase as a potential anti-malarial target: inhibition studies using improved methods for enzyme production and assay

ABSTRACT: BACKGROUND: There is an urgent need for the discovery of new anti-malarial drugs. Thus, it is essential to explore different potential new targets that are unique to the parasite or that are required for its viability in order to develop new interventions for treating the disease. Plasmodium serine hydroxymethyltransferase (SHMT), an enzyme in the dTMP synthesis cycle, is a potential target for such new drugs, but convenient methods for producing and assaying the enzyme are still lacking, hampering the ability to screen inhibitors. METHODS: Production of recombinant Plasmodium falciparum SHMT (PfSHMT) and Plasmodium vivax SHMT (PvSHMT), using auto-induction media, were compared to those using the conventional Luria Bertani medium with isopropyl thio-beta-D-galactoside (LB-IPTG) induction media. Plasmodium SHMT activity, kinetic parameters, and response to inhibitors were measured spectrophotometrically by coupling the reaction to that of 5,10-ethylenetetrahydrofolate dehydrogenase (MTHFD). The identity of the intermediate formed upon inactivation of Plasmodium SHMTs by thiosemicarbazide was investigated by spectrophotometry, high performance liquid chromatography (HPLC), and liquid chromatography-mass spectrometry (LC-MS). The active site environment of Plasmodium SHMT was probed based on changes in the fluorescence emission spectrum upon addition of amino acids and folate. RESULTS: Auto-induction media resulted in a two to three-fold higher yield of Pf- and PvSHMT (7.38 and 29.29 mg/L) compared to that produced in cells induced in LB-IPTG media. A convenient spectrophotometric activity assay coupling Plasmodium SHMT and MTHFD gave similar kinetic parameters to those previously obtained from the anaerobic assay coupling SHMT and 5,10-methylenetetrahydrofolate reductase (MTHFR); thus demonstrating the validity of the new assay procedure. The improved method was adopted to screen for Plasmodium SHMT inhibitors, of which some were originally designed as inhibitors of malarial dihydrofolate reductase. Plasmodium SHMT was slowly inactivated by thiosemicarbazide and formed a covalent intermediate, PLP-thiosemicarbazone. CONCLUSIONS: Auto-induction media offers a cost-effective method for the production of Plasmodium SHMTs and should be applicable for other Plasmodium enzymes. The SHMT-MTHFD coupled assay is equivalent to the SHMT-MTHFR coupled assay, but is more convenient for inhibitor screening and other studies of the enzyme. In addition to inhibitors of malarial SHMT, the development of species-specific, anti-SHMT inhibitors is plausible due to the presence of differential active sites on the Plasmodium enzymes.     

Advantages of a mechanistic codon substitution model for evolutionary analysis of protein-coding sequences

BACKGROUND: A mechanistic codon substitution model, in which each codon substitution rate is proportional to the product of a codon mutation rate and the average fixation probability depending on the type of amino acid replacement, has advantages over nucleotide, amino acid, and empirical codon substitution models in evolutionary analysis of protein-coding sequences. It can approximate a wide range of codon substitution processes. If no selection pressure on amino acids is taken into account, it will become equivalent to a nucleotide substitution model. If mutation rates are assumed not to depend on the codon type, then it will become essentially equivalent to an amino acid substitution model. Mutation at the nucleotide level and selection at the amino acid level can be separately evaluated. RESULTS: The present scheme for single nucleotide mutations is equivalent to the general time-reversible model, but multiple nucleotide changes in infinitesimal time are allowed. Selective constraints on the respective types of amino acid replacements are tailored to each gene in a linear function of a given estimate of selective constraints. Their good estimates are those calculated by maximizing the respective likelihoods of empirical amino acid or codon substitution frequency matrices. Akaike and Bayesian information criteria indicate that the present model performs far better than the other substitution models for all five phylogenetic trees of highly-divergent to highly-homologous sequences of chloroplast, mitochondrial, and nuclear genes. It is also shown that multiple nucleotide changes in infinitesimal time are significant in long branches, although they may be caused by compensatory substitutions or other mechanisms. The variation of selective constraint over sites fits the datasets significantly better than variable mutation rates, except for 10 slow-evolving nuclear genes of 10 mammals. An critical finding for phylogenetic analysis is that assuming variable mutation rates over sites lead to the overestimation of branch lengths.     

Serine hydroxymethyltransferase activity in Trypanosoma cruzi, Trypanosoma rangeli and American Leishmania spp

Serine hydroxymethyltransferase (SHMT) was studied in several American trypanosomatids, Trypanosoma cruzi epimastigotes displaying, in contrast with T. rangeli, high enzymatic activity. Several Leishmania spp. members, including L. braziliensis, L. mexicana and L. garnhami promastigotes, under identical assay conditions, showed low enzymatic activity. The T. cruzi and leishmanial enzymes presented several different kinetic properties, and thus apparent Km for THF was 0.30 mM for the trypanosomal SHMT vs 0.60 mM for the leishmanial enzyme, while the apparent Km for serine was 0.40 mM for trypanosomal SHMT vs 0.15 mM for leishmanial enzyme. There were significant variations in the specific activity of SHMT between the several different trypanosomatids strains studied, but the meaning of these results is not clear because they showed no correlation either with taxonomy or infectivity.     

Serine hydroxymethyltransferase from spinach leaf mitochondria. Purification and characterization

A mitochondrial serine hydroxymethyltransferase (EC 2.1.2.1) has for the first time been purified close to homogeneity from a photosynthetically active tissue, spinach ( Spinacea oleracea L. cv Viking II) leaves. The specific activity of the enzyme was 7.8 μmol (mg protein)⁻¹ min⁻¹ using L-serine as substrate. The enzyme was stable for at least 8 weeks at 4°C in the presence of folate. The pH optimum was at pH 8.5 where the enzyme had a K_m for L-serine of 0.9 m M . Carboxymethoxylamine was a strong competitive inhibitor with a K₁ of 1.4 μM. An absorption spectrum taken of the enzyme in the presence of glycine and tetrahydrofolate showed a peak at 492 nm, probably originating from a substrate-enzyme complex. The molecular weight obtained by gel filtration was 209 kDa. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the purified enzyme showed that the apparent molecular weight of the subunit was 53 kDa, indicating four subunits.     

Serine hydroxymethyltransferase: mechanism of the racemization and transamination of D- and L-alanine

The reaction specificity and stereochemical control of Escherichia coli serine hydroxymethyltransferase were investigated with D- and L-alanine as substrates. An active-site H228N mutant enzyme binds both D- and L-alanine with Kd values of 5 mM as compared to 30 and 10 mM, respectively, for the wild-type enzyme. Both wild-type and H228N enzymes form quinonoid complexes absorbing at 505 nm by catalyzing the loss of the alpha-proton from both D- and L-alanine. Racemization and transamination reactions were observed to occur with both alanine isomers as substrates. The relative rates of these reactions are quinonoid formation greater than alpha-proton solvent exchange greater than racemization greater than transamination. The observation that the rate of quinonoid formation with either alanine isomer is an order of magnitude faster than solvent exchange suggests that the alpha-protons from both D- and L-alanine are transferred to base(s) on the enzyme. The rate of racemization is 2 orders of magnitude slower than the formation of the quinonoid complexes. This latter difference in rate suggests that the quinonoid complexes formed from D- and L-alanine are not identical. The difference in structure of the two quinonoid complexes is proposed to be the active-site location of the alpha-protons lost from the two alanine isomers, rather than two orientations of the pyridoxal phosphate ring. The results are consistent with a two-base mechanism for racemization.     

Site-specific isotope labeling of long RNA for structural and mechanistic studies

A site-specific isotope labeling technique of long RNA molecules was established. This technique is comprised of two simple enzymatic reactions, namely a guanosine transfer reaction of group I self-splicing introns and a ligation with T4 DNA ligase. The trans-acting group I self-splicing intron with its external cofactor, 'isotopically labeled guanosine 5'-monophosphate' (5'-GMP), steadily gave a 5'-residue-labeled RNA fragment. This key reaction, in combination with a ligation of 5'-remainder non-labeled sequence, allowed us to prepare a site-specifically labeled RNA molecule in a high yield, and its production was confirmed with (15)N NMR spectroscopy. Such a site-specifically labeled RNA molecule can be used to detect a molecular interaction and to probe chemical features of catalytically/structurally important residues with NMR spectroscopy and possibly Raman spectroscopy and mass spectrometry.     

Enzyme promiscuity: evolutionary and mechanistic aspects

The past few years have seen significant advances in research related to the 'latent skills' of enzymes - namely, their capacity to promiscuously catalyze reactions other than the ones they evolved for. These advances regard (i) the mechanism of catalytic promiscuity - how enzymes, that generally exert exquisite specificity, promiscuously catalyze other, and sometimes barely related, reactions; (ii) the evolvability of promiscuous functions - namely, how latent activities evolve further, and in particular, how promiscuous activities can firstly evolve without severely compromising the original activity. These findings have interesting implications on our understanding of how new enzymes evolve. They support the key role of catalytic promiscuity in the natural history of enzymes, and suggest that today's enzymes diverged from ancestral proteins catalyzing a whole range of activities at low levels, to create families and superfamilies of potent and highly specialized enzymes.     

Informing phenomenological structural bone remodelling with a mechanistic poroelastic model

Studies suggest that fluid motion in the extracellular space may be involved in the cellular mechanosensitivity at play in the bone tissue adaptation process. Previously, the authors developed a mesoscale predictive structural model of the femur using truss elements to represent trabecular bone, relying on a phenomenological strain-based bone adaptation algorithm. In order to introduce a response to bending and shear, the authors considered the use of beam elements, requiring a new formulation of the bone adaptation drivers. The primary goal of the study presented here was to isolate phenomenological drivers based on the results of a mechanistic approach to be used with a beam element representation of trabecular bone in mesoscale structural modelling. A single-beam model and a microscale poroelastic model of a single trabecula were developed. A mechanistic iterative adaptation algorithm was implemented based on fluid motion velocity through the bone matrix pores to predict the remodelled geometries of the poroelastic trabecula under 42 different loading scenarios. Regression analyses were used to correlate the changes in poroelastic trabecula thickness and orientation to the initial strain outputs of the beam model. Linear (\(R^2>0.998\)) and third-order polynomial (\(R^2 >0.98\)) relationships were found between change in cross section and axial strain at the central axis, and between beam reorientation and ratio of bending strain to axial strain, respectively. Implementing these relationships into the phenomenological predictive algorithm for the mesoscale structural femur has the potential to produce a model combining biofidelic structure and mechanical behaviour with computational efficiency.     

Serine hydroxymethyltransferase: role of glu75 and evidence that serine is cleaved by a retroaldol mechanism

Serine hydroxymethyltransferase (SHMT) catalyzes the reversible interconversion of serine and glycine with tetrahydrofolate serving as the one-carbon carrier. SHMT also catalyzes the folate-independent retroaldol cleavage of allothreonine and 3-phenylserine and the irreversible conversion of 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate. Studies of wild-type and site mutants of SHMT have failed to clearly establish the mechanism of this enzyme. The cleavage of 3-hydroxy amino acids to glycine and an aldehyde occurs by a retroaldol mechanism. However, the folate-dependent cleavage of serine can be described by either the same retroaldol mechanism with formaldehyde as an enzyme-bound intermediate or by a nucleophilic displacement mechanism in which N5 of tetrahydrofolate displaces the C3 hydroxyl of serine, forming a covalent intermediate. Glu75 of SHMT is clearly involved in the reaction mechanism; it is within hydrogen bonding distance of the hydroxyl group of serine and the formyl group of 5-formyltetrahydrofolate in complexes of these species with SHMT. This residue was changed to Leu and Gln, and the structures, kinetics, and spectral properties of the site mutants were determined. Neither mutation significantly changed the structure of SHMT, the spectral properties of its complexes, or the kinetics of the retroaldol cleavage of allothreonine and 3-phenylserine. However, both mutations blocked the folate-dependent serine-to-glycine reaction and the conversion of methenyltetrahydrofolate to 5-formyltetrahydrofolate. These results clearly indicate that interaction of Glu75 with folate is required for folate-dependent reactions catalyzed by SHMT. Moreover, we can now propose a promising modification to the retroaldol mechanism for serine cleavage. As the first step, N5 of tetrahydrofolate makes a nucleophilic attack on C3 of serine, breaking the C2-C3 bond to form N5-hydroxymethylenetetrahydrofolate and an enzyme-bound glycine anion. The transient formation of formaldehyde as an intermediate is possible, but not required. This mechanism explains the greatly enhanced rate of serine cleavage in the presence of folate, and avoids some serious difficulties presented by the nucleophilic displacement mechanism involving breakage of the C3-OH bond.     

Polymorphism C1420T of Serine hydroxymethyltransferase gene on maternal risk for Down syndrome

Recent researches have investigated the factors that determine the maternal risk for Down syndrome (DS) in young woman. In this context, some studies have demonstrated the association between polymorphisms in genes involved on folate metabolism and the maternal risk for DS. These polymorphisms may result in abnormal folate metabolism and methyl deficiency, which is associated with aberrant chromosome segregation leading to trisomy 21. In this study, we analyzed the influence of the polymorphism C1420T in Serine hydroxymethyltransferase (SHMT) gene on maternal risk for DS and on metabolites concentrations of the folate pathway (serum folate and plasma homocysteine and methylmalonic acid). The study group was composed by 105 mothers with DS children (case group) and 185 mothers who had no children with DS (control group). The genotype distribution did not show significant statistical difference between case and control mothers (P?=?0.24) however a protective effect between genotypes CC (P?=?0.0002) and CT (P?<?0.0001) and maternal risk for DS was observed. Furthermore, the SHMT C1420T polymorphism (rs1979277) does not affect the concentration of metabolites of folate pathway in our DS mothers. In conclusion, our data showed a protective role for the genotypes SHMT CC and CT on maternal risk for DS. The concentrations of metabolites of folate pathway did not differ significantly between the genotypes SHMT.     

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.     

Gamma-glutamyl transferases: A structural,mechanistic and physiological perspective

Gamma glutamyl transferases （GGT） are highly conserved enzymes that occur from bacteria to humans. They remove terminal y-glutamyl residue from peptides and amides. GGTs play an important role in the homeostasis of glutathione （a major cellular antioxidant） and in the detoxification of xenobiotics in mammals. They are implicated in diseases like diabetes, inflammation, neurodegenerative diseases and cardiovascular diseases. The physiological role of GGTs in bacteria is still unclear. Nothing is known about the basis for the strong conservation of the enzyme across the living system. The review focuses on the enzyme＇s physiology, chemistry and structural properties of the enzyme with emphasis on the evolutionary relationships. The available data indicate that the members of the GGT family share common structural features but are functionally heterogenous.