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.     

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.     

Effect of vitamin B6 availability on serine hydroxymethyltransferase in MCF-7 cells

Folate-activated one-carbon units are derived from serine through the activity of the pyridoxal-phosphate (PLP)-dependent isozymes of serine hydroxymethyltransferase (SHMT). The effect of vitamin B(6) availability on the activity and expression of the human mitochondrial and cytoplasmic SHMT isozymes was investigated in human MCF-7 cells. Cells were cultured for 6 months in vitamin B(6) replete (4.9 microM pyridoxine) minimal essential medium (alphaMEM) or vitamin B(6)-deficient medium containing 49, 4.9 or 0.49 nM pyridoxine. Total cellular PLP levels and SHMT activity were reduced 72% and 7%, respectively, when medium pyridoxine was decreased from 4.9 microM to 49 nM. Cells cultured in medium containing 4.9 nM pyridoxine exhibited 75%, 27% and 60% reduced levels of PLP, SHMT activity and S-adenosylmethionine, respectively, compared to cells cultured in alphaMEM. Cytoplasmic SHMT activity and protein levels, but not mRNA levels, were decreased in cells cultured in vitamin B(6) deficient medium, whereas mitochondrial SHMT activity and protein levels were less sensitive to vitamin B(6) availability. PLP bound to cytoplasmic SHMT with a K(d)=850 nM, a value two orders of magnitude lower than previously reported for the bovine cytoplasmic SHMT isozyme. Collectively, these data indicate that vitamin B(6) restriction decreases the activity and stability of SHMT, and that the cytoplasmic isozyme is more sensitive to vitamin B(6) deficiency than the mitochondrial isozyme in MCF-7 cells.     

Molecular cloning and biochemical characterization of Leishmania donovani serine hydroxymethyltransferase

Serine hydroxymethyltransferase (SHMT) catalyzes the inter conversion of serine and tetrahydrofolate (H(4)-folate) to form glycine and 5,10-methylene H(4)-folate and generates one-carbon fragments for the synthesis of nucleotides, methionine, thymidylate, choline, etc. In spite of being an indispensable enzyme of the thymidylate cycle, SHMT in Leishmania donovani remains uncharacterized. The study of L. donovani SHMT (ldSHMT) becomes important as this gene is preferentially expressed in the amastigote stage of parasite, which resides in human macrophages. Here we report cloning, expression and purification of a catalytically active ldSHMT. The homogeneity of recombinant protein was analyzed by denaturing gel electrophoresis and protein was found to be 95% pure having yield of 1mg/l. The recombinant protein is a tetramer of 216kDa as evidenced by gel filtration chromatography and uses serine and tetrahydrofolate as substrates with Km of 1.6 and 2.4mM, respectively. Further biochemical studies revealed that pH optimum of ldSHMT is 7.8 and enzyme is thermally stable up to 45 degrees C. ldSHMT was found sensitive towards denaturants as manifested by loss of enzyme activity at the concentration of 1M urea or 0.25M guanidine hydrochloride. This is the first report of purification and characterization of recombinant SHMT from any protozoan source. Studies on recombinant ldSHMT will help in evaluating this enzyme as potential drug target.     

Purification,physicochemical and regulatory properties of serine hydroxymethyltransferase from sheep liver

Serine hydroxymethyltransferase (EC 2.1.2.1) was purified from the cytosolic fraction of sheep liver by ammonium sulphate fractionation, CM-Sephadex chromatography, gel filtration using Ultrogel ACA 34 and Blue Sepharose affinity chromatography. The homogeneity of the enzyme was rigorously established by Polyacrylamide gel and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, isoelectrofocusing, ultracentrifugation, immunodiffusion and Immunoelectrophoresis. The enzyme was a homotetramer with a molecular weight of 210,000 ±5000. The enzyme showed homotropic cooperative interactions with tetrahydrofolate (n_H =2.8) and a hyperbolic saturation pattern with L-serine. At the lowest concentration of tetrahydrofolate used (0.2 mM), only 5% of the added folate was oxidized during preincubation and assay. TheⁿH value was independent of the time of preincubation. Preincubation of the enzyme with serine resulted in a partial loss of the cooperative interactions (n_H =1.6) with tetrahydrofolate. The enzyme was regulated allosterically by interaction with nicotinamide nucleotides; NADH was a positive effector while NAD+ was a negative allosteric effector. The subunit interactions were retained even at the temperature optimum of 60‡C unlike in the case of the monkey liver enzyme, where these interactions were absent at higher temperatures. D-Cycloserine, a structural analogue of serine caused a sigmoid pattern of inhibition, in contrast with the observations on the monkey liver enzyme. Cibacron blue F3GA completely inhibited the enzyme and this inhibition could be reversed by tetrahydrofolate. Unlike in the monkey liver enzyme, NAD+ and NADH gave considerable protection against this inhibition. The sheep liver enzyme differs significantly in its kinetic and regulatory properties from the serine hydroxymethyltransferases isolated from other sources.     

Escherichia coli cis- and trans-acting mutations that increaseglyA gene expression

We used anEscherichia coli strain blocked in serine biosynthesis and carrying a partialglyA deletion to isolate strains with altered regulation of theglyA gene. TheglyA deletion results in 25% of the normal serine hydroxymethyltransferase activity. Three classes of mutants with increasedglyA expression were isolated on glycine supplemented plates. One class of mutations increasedglyA expression 10-fold by directly altering the – 35 consensus sequence of theglyA promoter. The two other classes increasedglyA expression about 2- and 6-fold, respectively. The latter two classes of mutations also affected regulation of themetE gene of the folate branch of the methionine pathway, but notmetA in the nonfolate branch of the methionine pathway, or thegcv operon, encoding the glycine cleavage enzyme system. The mutations were mapped to about minute 85.5 on theE. coli chromosome.     

Allosteric serine hydroxymethyltransferase from monkey liver: Correlation of conformational changes caused by denaturants with the alterations in catalytic activity

The far-ultraviolet region circular dichroic spectrumof serine hydroxymethyltransferase from monkey liver showed that the protein is in an α-helical conformation. The near ultraviolet circular dichoric spectrum revealed two negative bands originating from the tertiary conformational environment of the aromatic amino acid residues. Addition of urea or guanidinium chloride perturbed the characteristic fluorescence and far ultraviolet circular dichroic spectrum of the enzyme. The decrease in (θ)₂₂₂ and enzyme activity followed identical patterns with increasing concentrations of urea, whereas with guanidinium chloride, the loss of enzyme activity preceded the loss of secondary structure. 2-Chloroethanol, trifluoroethanol and sodium dodecyl sulphate enhanced the mean residue ellipticity values. In addition, sodium dodecyl sulphate also caused a perturbation of the fluorescence emission spectrum of the enzyme. Extremes of pH decreased the — (θ)₂₂₂ value. Plots of — (θ)₂₂₂and enzyme activity as a function of pH showed maximal values at pH 7.4–7.5. These results suggested the prevalence of “conformational flexibility” in the structure of serine hydroxymethyltransferase.     

Cloning, expression, and purification of 5,10-methenyltetrahydrofolate synthetase from Mus musculus

Folate metabolism is necessary for the biosyntheses of purine nucleotides and thymidylate and for the synthesis of S-adenosylmethionine, a cofactor required for cellular methylation reactions and a precursor of spermidine and spermine syntheses. Disruption of folate metabolism is associated with several pathologies and developmental anomalies including cancer and neural tube defects. The enzyme 5,10-methenyltetrahydrofolate synthetase (MTHFS, EC 6.3.3.2) catalyzes the ATP-dependent conversion of 5-formyltetrahydrofolate to 5,10-methenyltetrahydrofolate, and has been shown to affect intracellular folate concentrations by accelerating folate degradation. Mammalian MTHFS proteins described to date are not stable and no recombinant mammalian MTHFS protein has been successfully expressed in Escherichia coli. The three-dimensional structure of MTHFS has not been solved. The cDNA coding for Mus musculus MTHFS was isolated and expressed in E. coli with a hexa-histidine tag. Milligram quantities of recombinant mouse MTHFS were purified using metal affinity chromatography and the protein was stabilized with Tween 20. Mouse MTHFS has a molecular mass of 23 kDa and is 84% identical in amino acid sequence to the human enzyme. Activity assays confirmed the functionality of the recombinant protein, with K_m=5 μM for (6S)-5-formyltetrahydrofolate and K_m=769 μM for Mg–ATP. This is the first example of a mammalian form of MTHFS expressed in E. coli that yielded sufficient quantities of stable purified protein to allow for detailed characterization of its three-dimensional structure and kinetic properties.     

Enzymes of serine metabolism in normal and neoplastic rat tissues

Enzymes involved in the pathway of de novo serine biosynthesis (L-phosphoserine aminotransferase) and in alternative pathways of serine utilization (L-serine hydroxymethyltransferase, L-serine dehydratase and L-serine aminotransferase) were assayed in normal adult and fetal rat tissues and in a range of transplantable sat tumors. Serine dehydratase and serine aminotransferase activities were essentially confined to normal adult liver and kidney, whereas phosphoserine aminotransferase and serine hydroxymethyltransferase activities showed a more ubiquitous tissue distribution. In particular, phosphoserine aminotransferase and serine hydroxymethyltransferase activities were appreciable in neoplastic tissues, in the absence of the other enzymes of serine utilization. The pattern of enzyme distribution suggests that the synthesis of serine de novo is metabolically coupled to its utilization for nucleotide biosynthesis in tumors of differing tissue origins.     

Competition between sumoylation and ubiquitination of serine hydroxymethyltransferase 1 determines its nuclear localization and its accumulation in the nucleus

Serine hydroxymethyltransferase 1 (SHMT1) expression limits rates of de novo dTMP synthesis in the nucleus. Here we report that SHMT1 is ubiquitinated at the small ubiquitin-like modifier (SUMO) consensus motif and that ubiquitination at that site is required for SHMT1 degradation. SHMT1 protein levels are cell cycle-regulated, and Ub-SHMT1 levels are lowest at S phase when SHMT1 undergoes SUMO modification and nuclear transport. Mutation of the SUMO consensus motif increases SHMT1 stability. SHMT1 interacts with components of the proteasome in both the nucleus and cytoplasm, indicating that degradation occurs in both compartments. Ubc13-mediated ubiquitination is required for SHMT1 nuclear export and increases stability of SHMT1 within the nucleus, whereas Ubc9-mediated modification with Sumo2/3 is involved in nuclear degradation. These data demonstrate that SUMO and ubiquitin modification of SHMT1 occurs on the same lysine residue and determine the localization and accumulation of SHMT1 in the nucleus.     

Overexpression and characterization of dimeric and tetrameric forms of recombinant serine hydroxymethyltransferase from Bacillus stearothermophilus

Serine hydroxymethyltransferase (SHMT), a pyridoxal-5′-phosphate (PLP) dependent enzyme catalyzes the interconversion of L-Ser and Gly using tetrahydrofolate as a substrate. The gene encoding for SHMT was amplified by PCR from genomic DNA ofBacillus stearothermophilus and the PCR product was cloned and overexpressed inEscherichia coli. The purified recombinant enzyme was isolated as a mixture of dimer (90%) and tetramer (10%). This is the first report demonstrating the existence of SHMT as a dimer and tetramer in the same organism. The specific activities at 37°C of the dimeric and tetrameric forms were 6.7 U/mg and 4.1 U/mg, respectively. The purified dimer was extremely thermostable with aT _m of 85°C in the presence of PLP and L-Ser. The temperature optimum of the dimer was 80°C with a specific activity of 32.4 U/mg at this temperature. The enzyme catalyzed tetrahydrofolate-independent reactions at a slower rate compared to the tetrahydrofolate-dependent retro-aldol cleavage of L-Ser. The interaction with substrates and their analogues indicated that the orientation of PLP ring ofB. stearothermophilus SHMT was probably different from sheep liver cytosolic recombinant SHMT (scSHMT).     

Identification of a novel estrogen-sulfating cytosolic SULT from zebrafish: molecular cloning, expression, characterization, and ontogeny study

By searching the expressed sequence tag database, a zebrafish cDNA encoding a putative cytosolic sulfotransferase (SULT) was identified. Sequence analysis indicated that this zebrafish SULT belongs to the SULT1 cytosolic SULT gene family. The recombinant form of this novel zebrafish SULT, expressed using the pGEX-2TK expression system and purified from transformed BL21 (DE3) Escherichia coli cells, displayed sulfating activities specifically for estrone and 17beta-estradiol among various endogenous compounds tested as substrates. The enzyme also exhibited sulfating activities toward some xenobiotic phenolic compounds. This new zebrafish SULT showed dual pH optima, at 6.5 and 10-10.5, with estrone or n-propyl gallate as substrate. Kinetic constants of the sulfation of estrone, 17beta-estradiol, and n-propyl gallate were determined. Developmental stage-dependent expression experiments revealed a significant level of expression of this novel zebrafish estrogen-sulfating SULT at the beginning of the hatching period during embryogenesis, which continued throughout the larval stage onto maturity.     

A novel anti-plant viral protein from coelomic fluid of the earthworm Eisenia foetida: purification, characterization and its identification as a serine protease

A novel protein showing strong antiviral activities against cucumber mosaic virus (CMV) and tomato mosaic virus (TMV) was purified from the coelomic fluid of the earthworm Eisenia foetida. The protein was characterized as a cold-adapted serine protease. Its molecular weight was estimated to be 27,000 by SDS-PAGE. The enzyme was most active at pH 9.5 and 40–50 °C. The protease activity at 4 °C was 60% of that obtained at the optimal temperature. The activity was suppressed by various serine protease inhibitors. Partial N-terminal amino acid sequence of the enzyme showed homology with serine proteases of earthworms, E. foetida and Lumbricus rubellus previously studied. Our results suggest that the enzyme can be applicable as a potential antiviral factor against CMV, TMV, and other plant viruses.     

Cloning and characterization of the gene for Salmonella typhimurium serine hydroxymethyltransferase

A plasmid containing the glyA gene of Salmonella typhimurium LT2 was constructed in vitro using plasmid pACYC184 as the cloning vector and a λgt7-glyA transducing phage as the source of glyA DNA. The recombinant plasmid (pGS30) contains a 10-kb EcoRI insert fragment. Genetic and biochemical experiments established that the fragment contains a functional glyA gene. From plasmid pGS30 we subcloned a 4.4-kb SalI-EcoRI fragment containing the glyA gene and its neighboring regions (plasmid pGS38). The location and orientation of the glyA gene within the 4.4-kb insert fragment was determined in four ways: (1) comparison of the physical map of the 4.4-kb SalI-EcoRI fragment with the physical map of a 2.6-kb SalI-PvuII fragment that carries the Escherichia coli glyA gene; (2) deletion analysis; (3) transposon Tn5 insertional inactivation experiments; (4) deoxyribonucleic acid sequencing and comparison of the S. typhimurium DNA sequence with the E. coli DNA sequence. A presumptive glyA-encoded polypeptide of M_r 47000 was detected using plasmid pGS38 as template in a minicell system, but not when the glyA gene was inactivated by insertion of a Tn5 element.