Progress in using threonine aldolases for preparative synthesis.

Three threonine aldolases (TAs) were cloned and overexpressed in Escherichia coli (Aeromonas jandaeil-allo-threonine aldolase, E. colil-threonine aldolase and Thermotoga maritimal-allo-threonine aldolase). A Design of Experiments strategy was used to identify optimal reaction conditions for each enzyme. These conditions were used to characterize the substrate- and stereoselectivity of each TA toward a panel of aldehyde acceptors. In general, the A. jandaei TA performed best, and six representative conversions were scaled up 10-fold in order to develop downstream steps for product isolation. One key improvement was to treat the crude reaction product with Bacillus subtilis glycine oxidase, which eliminated residual starting material and significantly simplified product isolation. NMR studies were used to identify the major and minor diastereomers from the preparative-scale reactions and the absolute configurations for three representative cases.

Isolation and functional characterization of 3-phosphoglycerate dehydrogenase involved in salt responses in sugar beet.

The present study investigated the significance of serine biosynthetic genes for salt stress in sugar beet (Beta vulgaris). We isolated a total of four genes, two each encoding D-3-phosphoglycerate dehydrogenase (BvPGDHa and BvPGDHb) and serine hydroxymethyl transferase (BvSHMTa and BvSHMTb). mRNA transcriptional expression for BvPGDHa was significantly enhanced under salt stress conditions in both leaves and roots of sugar beet, whereas it was reduced for BvPGDHb. On the other hand, BvSHMTa was expressed transiently in leaves and roots under salt stress, whereas expression level of BvSHMTb was not altered. PGDH activity was high in storage root. After salt stress, PGDH activity was increased in leaf, petiole, and root. Recombinant proteins were expressed in Escherichia coli. The K m values for 3-phosphoglycerate in PGDHa and PGDHb were 1.38 and 2.92 mM, respectively. The findings suggest that BvPGDHa and BvSHMTa play an important role during salt stress in sugar beet.

Isolation of Arabidopsis Leaf Peroxisomes and the Peroxisomal Membrane.

To date, less than 150 proteins have been located to plant peroxisomes, indicating that unbiased large-scale approaches such as experimental proteome research are required to uncover the remaining yet unknown metabolic functions of this organelle as well as its regulatory mechanisms and membrane proteins. For experimental proteome research, Arabidopsis thaliana is the model plant of choice and an isolation methodology that obtains peroxisomes of sufficient yield and high purity is vital for research on this organelle. However, organelle enrichment is more difficult from Arabidopsis when compared to other plant species and especially challenging for peroxisomes. Leaf peroxisomes from Arabidopsis are very fragile in aqueous solution and show pronounced physical interactions with chloroplasts and mitochondria in vivo that persist in vitro and decrease peroxisome purity. Here, we provide a detailed protocol for the isolation of Arabidopsis leaf peroxisomes using two different types of density gradients (Percoll and sucrose) sequentially that yields approximately 120 µg of peroxisome proteins from 60 g of fresh leaf material. A method is also provided to assess the relative purity of the isolated peroxisomes by immunoblotting to allow selection of the purest peroxisome isolates. To enable the analysis of peroxisomal membrane proteins, an enrichment strategy using sodium carbonate treatment of isolated peroxisome membranes has been adapted to suit isolated leaf peroxisomes and is described here.

Development of a growth-dependent selection system for identification of L-threonine aldolases.

Threonine aldolases (TAs) are useful enzymes for the synthesis of ß-hydroxy-α-amino acids due to their capability to catalyze asymmetric aldol reactions. Starting from two prochiral compounds, an aldehyde and glycine, two chiral stereocenters were formed in a single step via C-C bond formation. Owing to poor diastereoselectivity and low activity, the enzymatic synthesis of ß-hydroxy-α-amino acids by TAs is still a challenge. For identification of new TAs, a growth-dependent selection system in Pseudomonas putida KT2440 has been developed. This bacterium is able to use aromatic compounds such as benzaldehyde, which is the cleavage product of the TA-mediated retro-aldol reaction of phenylserine, as sole carbon source via the ß-ketoadipate pathway. With DL-threo-ß-phenylserine as sole carbon source, this strain showed only slight growth in minimal medium. This growth deficiency can be restored by introducing and expressing genes encoding TAs. In order to develop a highly efficient selection system, the gene taPp of P. putida KT2440 encoding a TA was successfully deleted by replacement with an antibiotic resistance cassette. Different growth studies were carried out to prove the operability of the selection system. Genes encoding for L- and D-specific TAs (L-TA genes of Escherichia coli (ltaE) and Saccharomyces cerevisiae (gly1) and D-TA gene of Achromobacter xylosoxidans (dtaAX)) were introduced into the selection strain P. putida KT2440ΔtaPp, followed by cultivation on minimal medium supplemented with DL-threo-ß-phenylserine. The results demonstrate that only the selection strains with plasmid-encoded L-TAs were able to grow on this racemic amino acid, whereas the corresponding strain harboring the gene coding for a D-specific TA showed no growth. In summary, it can be stated that a powerful screening tool was developed to identify easily by growth new L-specific threonine aldolases or other enzymes from genomic or metagenomic libraries liberating benzaldehyde.

Characterization of serine hydroxymethyltransferase GlyA as a potential source of D-alanine in Chlamydia pneumoniae.

For intracellular Chlamydiaceae, there is no need to withstand osmotic challenges, and a functional cell wall has not been detected in these pathogens so far. Nevertheless, penicillin inhibits cell division in Chlamydiaceae resulting in enlarged aberrant bodies, a phenomenon known as chlamydial anomaly. D-alanine is a unique and essential component in the biosynthesis of bacterial cell walls. In free-living bacteria like Escherichia coli, penicillin-binding proteins such as monofunctional transpeptidases PBP2 and PBP3, the putative targets of penicillin in Chlamydiaceae, cross-link adjacent peptidoglycan strands via meso-diaminopimelic acid and D-Ala-D-Ala moieties of pentapeptide side chains. In the absence of genes coding for alanine racemase Alr and DadX homologs, the source of D-Ala and thus the presence of substrates for PBP2 and PBP3 activity in Chlamydiaceae has puzzled researchers for years. Interestingly, Chlamydiaceae genomes encode GlyA, a serine hydroxymethyltransferase that has been shown to exhibit slow racemization of D- and L-alanine as a side reaction in E. coli. We show that GlyA from Chlamydia pneumoniae can serve as a source of D-Ala. GlyA partially reversed the D-Ala auxotrophic phenotype of an E. coli racemase double mutant. Moreover, purified chlamydial GlyA had racemase activity on L-Ala in vitro and was inhibited by D-cycloserine, identifying GlyA, besides D-Ala ligase MurC/Ddl, as an additional target of this competitive inhibitor in Chlamydiaceae. Proof of D-Ala biosynthesis in Chlamydiaceae helps to clarify the structure of cell wall precursor lipid II and the role of chlamydial penicillin-binding proteins in the development of non-dividing aberrant chlamydial bodies and persistence in the presence of penicillin.

A serine hydroxymethyltransferase from marine bacterium Shewanella algae: Isolation, purification, characterization and l-serine production.

Currently, l-serine is mainly produced by enzymatic conversion, in which serine hydroxymethyltransferase (SHMT) is the key enzyme, suggesting the importance of searching for a SHMT with high activity. Shewanella algae, a methanol-utilizing marine bacterium showing high SHMT activity, was selected based on screening bacterial strains and comparison of the activities of SHMTs. A glyA was isolated from the S. algae through thermal asymmetric interlaced PCR (TAIL-PCR) and it encoded a 417 amino acid polypeptide. The SaSHMT, encoded by the glyA, showed the optimal activity at 50°C and pH 7.0, and retained over 45% of its maximal activity after incubation at 40°C for 3h. The enzyme showed better stability under alkaline environment (pH 6.5-9.0) than Hyphomicrobium methylovorum GM2's SHMT (pH 6.0-7.5). The SaSHMT can produce 77.76mM of l-serine by enzymatic conversion, with the molecular conversion rate in catalyzing glycine to l-serine being 1.41-fold higher than that of Escherichia coli. Therefore, the SaSHMT has the potential for industrial applications due to its tolerance of alkaline environment and a relatively high enzymatic conversion rate.

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

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-ß-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-methylenetetrahydrofolate 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.

Serine hydroxymethyltransferase from the cold adapted microorganism Psychromonas ingrahamii: a low temperature active enzyme with broad substrate specificity.

Serine hydroxymethyltransferase from the psychrophilic microorganism Psychromonas ingrahamii was expressed in Escherichia coli and purified as a His-tag fusion protein. The enzyme was characterized with respect to its spectroscopic, catalytic, and thermodynamic properties. The properties of the psychrophilic enzyme have been contrasted with the characteristics of the homologous counterpart from E. coli, which has been structurally and functionally characterized in depth and with which it shares 75% sequence identity. Spectroscopic measures confirmed that the psychrophilic enzyme displays structural properties almost identical to those of the mesophilic counterpart. At variance, the P. ingrahamii enzyme showed decreased thermostability and high specific activity at low temperature, both of which are typical features of cold adapted enzymes. Furthermore, it was a more efficient biocatalyst compared to E. coli serine hydroxymethyltransferase (SHMT) particularly for side reactions. Many ß-hydroxy-α-amino acids are SHMT substrates and represent important compounds in the synthesis of pharmaceuticals, agrochemicals and food additives. Thanks to these attractive properties, this enzyme could have a significant potential for biotechnological applications.

In silico and in vitro validation of serine hydroxymethyltransferase as a chemotherapeutic target of the antifolate drug pemetrexed.

Serine hydroxymethyltransferase (SHMT), a ubiquitous representative of the family of fold-type I, pyridoxal 5'-phosphate (PLP) dependent enzymes, catalyzes the reversible conversion of tetrahydrofolate (H4PteGlu) and serine to 5,10-CH2-H4PteGlu and glycine. Together with thymidylate synthase (TS) and dihydrofolate reductase (DHFR), SHMT participates to the thymidylate (dTMP) biosynthetic process. Elevated SHMT activity has been coupled to the increased demand for DNA synthesis in tumour cells. However, SHMT is the only enzyme of the thymidylate cycle yet to be targeted by chemotherapeutics. In this study, the interaction mode of SHMT with pemetrexed, an antifolate drug inhibiting several enzymes involved in folate-dependent biosynthetic pathways, was assessed. The mechanism of SHMT inhibition by pemetrexed was investigated in vitro using the human recombinant protein. The results of this study showed that pemetrexed competitively inhibits SHMT with respect to H4PteGlu with a measured Ki of 19.1±3.1 µM; this value was consistent with a Kd of 16.9±5.0 µM, measured by isothermal titration calorimetry. The binding mode of pemetrexed to SHMT was further investigated by molecular docking. The calculated interaction energy of pemetrexed in the active site of SHMT was -7.48 kcal/mol, and the corresponding predicted binding affinity was 36.3 µM, in good agreement with Kd and Ki values determined experimentally. The results thus provide insights into the mechanism of action of this antifolate drug and constitute the basis for the rational design of more selective inhibitors of SHMT.

Threonine aldolases-screening, properties and applications in the synthesis of non-proteinogenic beta-hydroxy-alpha-amino acids.

Threonine aldolases (TAs) constitute a powerful tool for catalyzing carbon-carbon bond formations in synthetic organic chemistry, thus enabling an enantio- and diastereoselective synthesis of beta-hydroxy-alpha-amino acids. Starting from the achiral precursors glycine and an aldehyde, two new stereogenic centres are formed in this catalytic step. The resulting chiral beta-hydroxy-alpha-amino acid products are important precursors for pharmaceuticals such as thiamphenicol, a L: -threo-phenylserine derivative or L: -threo-3,4-dihydroxyphenylserine. TAs are pyridoxal-5-phosphate-dependent enzymes, which, in nature, catalyze the cleavage of L: -threonine or L: -allo-threonine to glycine and acetaldehyde in a glycine biosynthetic pathway. TAs from a broad number of species of bacteria and fungi have been isolated and characterised as biocatalysts for the synthesis of beta-hydroxy-alpha-amino acids. In this review, screening methods to obtain novel TAs, their biological function, biochemical characterisation and preparative biotransformations with TAs are described.

Enhancing production of L-serine by increasing the glyA gene expression in Methylobacterium sp. MB200.

Microbial fermentation using methylotrophic bacteria is one of the most promising methods for L-serine production. Here we describe the metabolic engineering of a Methylobacterium strain to increase the production of L-serine. The glyA gene, encoding serine hydroxymethyltransferase (SHMT), was isolated from the genomic DNA of Methylobacterium sp. MB200, using a DNA fragment encoding Methylobacterium extorquens AM1 SHMT as a probe, and inserted into the vector pLAFR3. The resulting construct was transformed into Methylobacterium sp. MB200 using triparental mating. The genetic-engineered strain, designated as Methylobacterium sp. MB202, was shown to produce 11.4 + or - 0.6 mg/ml serine in resting cell reactions from 30 mg/ml wet cells, 20 mg/ml glycine, and 70 mg/ml methanol in 2 days, representing a 4.4-fold increase from that of the wild strain. The results demonstrated the potential for improving L-serine production by manipulating the glyA in bacteria and should facilitate the production of L-serine using Methylobacterium sp. strains.

Characterization of Plasmodium falciparum serine hydroxymethyltransferase-A potential antimalarial target.

Serine hydroxymethyltransferase (SHMT) is a ubiquitous enzyme required for folate recycling and dTMP synthesis. A cDNA encoding Plasmodium falciparum (Pf) SHMT was expressed as a hexa-histidine tagged protein in Escherichia coli BL21-CodonPlus (DE3)-RIL. The protein was purified and the process yielded 3.6 mg protein/l cell culture. Recombinant His(6)-tagged PfSHMT exhibits a visible spectrum characteristic of pyridoxal-5'-phosphate enzyme and catalyzes the reversible conversion of l-serine and tetrahydrofolate (H(4)folate) to glycine and 5,10-methylenetetrahydrofolate (CH(2)-H(4)folate). Steady-state kinetics study indicates that His(6)-tagged PfSHMT catalyzes the reaction by a ternary-complex mechanism. The sequence of substrate binding to the enzyme was also examined by glycine product inhibition. A striking property that is unique for His(6)-tagged PfSHMT is the ability to use D-serine as a substrate in the folate-dependent serine-glycine conversion. Kinetic data in combination with expression result support the proposal of SHMT reaction being a regulatory step for dTMP cycle. This finding suggests that PfSHMT can be a potential target for antimalarial chemotherapy.

Catalytic and ligand-binding characteristics of Plasmodium falciparum serine hydroxymethyltransferase.

The plant-like, bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) from malaria parasites has been a good target for drug development. Dihydrofolate reductase (DHFR) is inhibited by clinically established antimalarials, pyrimethamine and cycloguanil. Thymidylate synthase (TS) is the target of potent experimental antimalarials such as 5-fluoroorotate and 1843U89. Another enzyme in folate recycling, serine hydroxymethyltransferase (SHMT), produces 5,10-methylenetetrahydrofolate which, in many cells, is required for the de novo, biosynthesis of thymidine and methionine. Thus, the biochemical characterization of malarial SHMT was of interest. The principle, active Plasmodium falciparum SHMT (PfSHMT) was expressed in E. coli and purified using an N-terminal histidine tag. Unlike the plant enzyme, but like the host enzyme, PfSHMT requires the cofactor pyridoxal 5'-phosphate for enzymatic activity. The substrate specificities for serine, tetrahydrofolate, and pyridoxal 5'-phosphate were comparable to those for SHMT from other organisms. Antifolates developed for DHFR and TS inhibited SHMT in the mid-micromolar range, offering insights into the binding preferences of SHMT but clearly leaving room for improved new inhibitors. As previously seen with P. falciparum DHFR-TS, PfSHMT bound its cognate mRNA but not control RNA for actin. RNA binding was not reversed with enzyme substrates. Unlike DHFR-TS, the SHMT RNA-protein interaction was not tight enough to inhibit translation. Another gene PF14_0534, previously proposed to code for an alternate mitochondrial SHMT, was also expressed in E. coli but found to be inactive. This protein, nor DHFR-TS, enhanced the catalytic activity of PfSHMT. The present results set the stage for developing specific, potent inhibitors of SHMT from P. falciparum.

Serine hydroxymethyltransferase from Plasmodium vivax is different in substrate specificity from its homologues.

The putative gene of Plasmodium vivax serine hydroxymethyltransferase (PvSHMT; EC 2.1.2.1) was cloned and expressed in Escherichia coli. The purified enzyme was shown to be a dimeric protein with a monomeric molecular mass of 49 kDa. PvSHMT has a maximum absorption peak at 422 nm with a molar absorption coefficient of 6370 M(-1) x cm(-1). The K(d) for binding of the enzyme and pyridoxal-5-phosphate was 0.14 +/- 0.01 microM. An alternative assay for measuring the tetrahydrofolate-dependent SHMT activity based on the coupled reaction with 5,10-methylenetetrahydrofolate reductase (EC 1.5.1.20) from E. coli was developed. PvSHMT uses a ternary-complex mechanism with a k(cat) value of 0.98 +/- 0.06 s(-1) and K(m) values of 0.18 +/- 0.03 and 0.14 +/- 0.02 mM for L-serine and tetrahydrofolate, respectively. The optimum pH of the SHMT reaction was 8.0 and an Arrhenius's plot showed a transition temperature of 19 degrees C. Besides L-serine, PvSHMT forms an external aldimine complex with D-serine, L-alanine, L-threonine and glycine. PvSHMT also catalyzes the tetrahydrofolate-independent retro-aldol cleavage of 3-hydroxy amino acids. Although L-serine is a physiological substrate for SHMT in the tetrahydrofolate-dependent reaction, PvSHMT can also use D-serine. In the absence of tetrahydrofolate at high pH, PvSHMT forms an enzyme-quinonoid complex with D-serine, but not with L-serine, whereas SHMT from rabbit liver was reported to form an enzyme-quinonoid complex with L-serine. The substrate specificity difference between PvSHMT and the mammalian enzyme indicates the dissimilarity between their active sites, which could be exploited for the development of specific inhibitors against PvSHMT.

Cloning of the gene encoding alpha-methylserine hydroxymethyltransferase from Aminobacter sp. AJ110403 and Ensifer sp. AJ110404 and characterization of the recombinant enzyme.

Genes encoding alpha-methylserine hydroxymethyltransferase from Aminobacter sp. AJ110403 and Ensifer sp. AJ110404 were cloned and expressed in Escherichia coli. The purified enzymes were homodimers with a 46-kDa subunit and contained 1 mol/mol-subunit of pyridoxal 5'-phosphate. The V(max) of these enzymes catalyzing the conversion of alpha-methyl-L-serine to D-alanine via tetrahydrofolate was 22.1 U/mg (AJ110403) and 15.4 U/mg (AJ110404).

Synthesis of L-threo-3,4-dihydroxyphenylserine (L-threo-DOPS) with thermostabilized low-specific L-threonine aldolase from Streptomyces coelicolor A3(2).

Stability-enhanced mutants, H44, 11-94, 5A2-84, and F8, of L-threonine aldolase (L-TA) from Streptomyces coelicolor A3(2) (SCO1085) were isolated by an error-prone PCR followed by a high-throughput screening. Each of these mutant, had a single amino acid substitution: H177Y in the H44 mutant, A169T in the 11-94 mutant, D104N in the 5A2-84 mutant and Fl81 in the F8 mutant. The residual L-TA activity of the wild-type L-TA after a heat treatment for 20 min at 60 degrees C was only 10.6%. However, those in the stability-enhanced mutants were 85.7% for the H44 mutant, 58.6% for the F8 mutant, 62.1% for the 5A2-84 mutant, and 67.6% for the 11-94 mutant. Although the half-life of the wild-type L-TA at 63 degrees C was 1.3 min, those of the mutant L-TAs were longer: 14.6 min for the H44 mutant, 3.7 min for the 11-94 mutant, 5.8 min for the 5A2-84 mutant, and 5.0 min for the F8 mutant. The specific activity did not change in most of the mutants, but it was decreased by 45% in the case of mutant F8. When the aldol condensation of glycine and 3,4-dihydroxybenzaldehyde was studied by using whole cells of Escherichia coli containing the wild-type L-TA gene, L-threo-3,4-dihydroxyphenylserine (L.-threo-DOPS) was successfully synthesized with a yield of 2.0 mg/ml after 20 repeated batch reactions for 100 h. However, the L-threo-DOPS synthesizing activity of the enzyme decreased with increased cycles of the batch reactions. Compared with the wild-type L-TA, H44 L-TA kept its L-threo-DOPS synthesizing activity almost constant during the 20 repeated batch reactions for 100 h, yielding 4.0 mg/ml of L-threo-DOPS. This result showed that H44 L-TA is more effective than the wild-type L-TA for the mass production of L-threo-DOPS.

Cloning, expression, purification, and characterization of zebrafish cytosolic serine hydroxymethyltransferase.

A cDNA which encodes for zebrafish serine hydroxymethyltransferase (SHMT) has been cloned into a pET43.1a vector as a NdeI-EcoRI insert and transformed into HMS174(DE3) cells. After induction with isopropyl thiogalactoside, the enzyme was purified with a three-step purification protocol and about 15 mg of pure enzyme was obtained per liter of culture. Spectral and structural characteristics of the recombinant zebrafish SHMT are similar to the rabbit and human cytosolic SHMT. Kinetic constants for the natural substrates l-serine and tetrahydrofolate are also comparable to the values obtained previously for the rabbit and human cytosolic enzyme.

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.

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 of Bacillus stearothermophilus and the PCR product was cloned and overexpressed in Escherichia 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 a T(m) of 85 degrees C in the presence of PLP and L-Ser. The temperature optimum of the dimer was 80 degrees 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 of B. stearothermophilus SHMT was probably different from sheep liver cytosolic recombinant SHMT (scSHMT).

Identification of glyA (encoding serine hydroxymethyltransferase) and its use together with the exporter ThrE to increase L-threonine accumulation by Corynebacterium glutamicum.

L-threonine can be made by the amino acid-producing bacterium Corynebacterium glutamicum. However, in the course of this process, some of the L-threonine is degraded to glycine. We detected an aldole cleavage activity of L-threonine in crude extracts with an activity of 2.2 nmol min(-1) (mg of protein)(-1). In order to discover the molecular reason for this activity, we cloned glyA, encoding serine hydroxymethyltransferase (SHMT). By using affinity-tagged glyA, SHMT was isolated and its substrate specificity was determined. The aldole cleavage activity of purified SHMT with L-threonine as the substrate was 1.3 micromol min(-1) (mg of protein)(-1), which was 4% of that with L-serine as substrate. Reduction of SHMT activity in vivo was obtained by placing the essential glyA gene in the chromosome under the control of P(tac), making glyA expression isopropylthiogalactopyranoside dependent. In this way, the SHMT activity in an L-threonine producer was reduced to 8% of the initial activity, which led to a 41% reduction in glycine, while L-threonine was simultaneously increased by 49%. The intracellular availability of L-threonine to aldole cleavage was also reduced by overexpressing the L-threonine exporter thrE. In C. glutamicum DR-17, which overexpresses thrE, accumulation of 67 mM instead of 49 mM L-threonine was obtained. This shows that the potential for amino acid formation can be considerably improved by reducing its intracellular degradation and increasing its export.