The presence of two serine racemases in Streptomyces garyphalus,a d-cycloserine producer

Two serine racemases (I and II) were isolated from Streptomyces garyphalus. Serine racemase I (molecular weight 93,000) was purified to a single band in an analytical electrofocusing system. Serine racemase II (molecular weight 73,000) was partially purified. Both enzymes used pyridoxal-5-phosphate as cofactor. Besides serine the enzymes utilized alanine as substrate but no other amino acid tested. The K _m values of l-alanine and l-serine for enzyme I were 111 mM and 35 mM respectively. Enzyme I was not inhibited by d-cycloserine but by hydroxylamine. Both substances inhibited enzyme II. The serine racemases may be involved in the biosynthesis of d-cycloserine in S. garyphalus.     

Characterization of prolidase I and II purified from normal human erythrocytes: comparison with prolidase in erythrocytes from a patient with prolidase deficiency

The effect of various sulfur-containing amino acids on the activities of prolidase isoenzymes I and II isolated from erythrocytes of healthy individuals, and erythrocyte lysates from a patient with prolidase deficiency was investigated. The activity of prolidase I against glycylproline was strongly enhanced by d-methionine. l-Methionine and d,l-methionine slightly enhanced the activity at low concentration, but N-acetyl-l-methionine had no effect. d-Ethionine, l-ethionine, and d,l-ethionine also enhanced the activity of prolidase I. d,l-Homocysteine enhanced the activity at low concentration, but inhibited the activity at 50 mM. The activity of prolidase II against methionylproline was enhanced by d-methionine, d,l-methionine, and l-methionine, but N-acetyl-l-methionine had no effect. d-Ethionine and d,l-ethionine strongly enhanced the activity of prolidase II compared with l-ethionine; d,l-homocysteine weakly enhanced the activity. d,l-Homocysteine-thiolactone inhibited the activities of prolidase I and II in a concentration-dependent manner. The effect of various sulfur-containing amino acids on prolidase activity against methionylproline in erythrocyte lysates from a patient with prolidase deficiency was almost the same as that on prolidase II. The kinetics of the activities of prolidase I, II, and patient prolidase were also studied. Their K _m values were changed by adding sulfur-containing amino acids, but V _max values were unchanged.     

Dominance of iminopeptidase activity in the human oral bacteriumTreponema denticola ATCC 35405

Treponema denticola ATCC 35405, a human oral spirochete associated with periodontal disease, was shown to contain three enzymes (I, II, and III) with proline iminopeptidase activity. II and III were considered to be true iminopeptidases, whereas enzyme I was found to be a benzoylarginine peptidase with iminopeptidase activity. Enzyme III, the dominant proline iminopeptidase ofT. denticola in terms of its activity towardN-l-prolyl-2-naphthylamine, was considered to be a sulfhydryl peptidase: 0.167 M p-chloromercuribenzoic acid totally inactivated the enzyme, and 1.0 mM dithiothreitol restored 92% of activity. The activity of this enzyme was not affected by metal chelators. Chemical modification of enzyme III suggests that tyrosyl (or histidyl) and carboxyl groups may be necessary for its activity. The hydrolysis ofN-l-prolyl-2-naphthylamine was found to be very characteristic ofT. denticola ATCC 35405; out of 24 differentN-l-aminoacyl-2-naphthylamines tested, only the proline derivative was hydrolyzed at a high rate. The substrate specificity of the enzymes discovered indicates that they may be important for the nutrition ofT. denticola. The iminopeptidase activity may be related to the pathogenicity of this organism in periodontal disease.     

Purification and characterization of intracellular α-l-rhamnosidase from Pseudomonas paucimobilis FP2001

α-l-Rhamnosidase was extracted and purified from the cells of Pseudomonas paucimobilis FP2001 with a 19.5% yield. The purified enzyme, which was homogeneous as shown by SDS-PAGE and isoelectric focusing, had a molecular weight of 112,000 and an isoelectric point of 7.1. The enzyme activity was accelerated by Ca²⁺ and remained stable for several months when stored at –20 °C. The optimum pH was 7.8; the optimum temperature was 45 °C. The K _m, V _max and k _cat for p-nitrophenyl α-l-rhamnopyranoside were 1.18 mM, 92.4 μM · min^–1 and 117,000 · min^–1, respectively. Examination of the substrate specificity using various synthetic and natural l-rhamnosyl glycosides showed that this enzyme had a relatively broader substrate specificity than those reported so far. Received: 24 May 1999 / Accepted: 7 October 1999     

Properties of Laccases Produced by Pycnoporus sanguineus Induced by 2,5-xylidine

Two isoforms of laccase produced from the culture supernatant of Pycnoporus sanguineus were partially purified by phenyl-Sepharose chromatography. Molecular masses of the enzymes were 80 kDa (Lac I) and 68 kDa (Lac II). Optimum activity of Lac I was at pH 4.8 and 30 °C, and Lac II was at pH 4.2 and 50 °C over 5 min reaction. The K_m values of enzymes toward syringaldazine were 10 μm (Lac I) and 8 μm (Lac II). Sodium azide inhibited Lac I (85%) and Lac II (75%) activities. Revisions requested 30 November 2005; Revisions received 26 January 2006     

Purification and characterization of 5-oxo-l-prolinase from Paecilomyces varioti F-1, an ATP-dependent hydrolase active with l-2-oxothiazolidine-4-carboxylic acid

An enzyme cleaving l-2-oxothiazolidine-4-carboxylic acid to l-cysteine was purified 75-fold with 8% recovery to near homogeneity from crude extracts of Paecilomyces varioti F-1, which had been isolated as a fungus able to assimilate l-2-oxothiazolidine-4-carboxylic acid. The molecular mass was estimated to be 260 kDa by gel filtration. The purified preparation migrated as a single band of molecular mass 140 kDa upon SDS-PAGE. The maximum activity was observed at a range of pH 7.0–8.0 and at 50 °C. The enzyme activity was completely inhibited by SH-blocking reagents such as AgNO₃, p-chloromercuribenzoic acid, N-ethylmaleimide, and N-bromosuccinimide. The enzyme required ATP, Mg²⁺, and KCl for the cleavage of l-2-oxothiazolidine-4-carboxylic acid. The enzyme also cleaved 5-oxo-l-proline to l-glutamic acid and is considered to be 5-oxo-l-prolinase. Received: 23 March 1999 / Accepted: 22 June 1999     

Purification and characterization of a high molecular mass serine carboxypeptidase from <Emphasis Type="Italic">Monascus pilosus</Emphasis>

Two serine carboxypeptidases, MpiCP-1 and MpiCP-2, were purified to homogeneity from Monascus pilosus IFO 4480. MpiCP-1 is a homodimer with a native molecular mass of 125 kDa composed of two identical subunits of 61 kDa, while MpiCP-2 is a high mass homooligomer with a native molecular mass of 2,263 kDa composed of about 38 identical subunits of 59 kDa. This is unique among carboxypeptidases and distinguishes MpiCP-2 as the largest known carboxypeptidase. The two purified enzymes were both acidic glycoproteins. MpiCP-1 has an isoelectric point of 3.7 and a carbohydrate content of 11%, while for MpiCP-2 these values were 4.0 and 33%, respectively. The optimum pH and temperature were around 4.0 and 50°C for MpiCP-1, and 3.5 and 50°C for MpiCP-2. MpiCP-1 was stable over a broad range of pH between 2.0 and 8.0 at 37°C for 1 h, and up to 55°C for 15 min at pH 6.0, but MpiCP-2 was stable in a narrow range of pH between 5.5 and 6.5, and up to 50°C for 15 min at pH 6.0. Phenylmethylsulfonylfluoride strongly inhibited MpiCP-1 and completely inhibited MpiCP-2, suggesting that they are both serine carboxypeptidases. Of the substrates tested, benzyloxycarbonyl-l-tyrosyl-l-glutamic acid (Z-Tyr-Glu) was the best for both enzymes. The K_m, V_max, K_cat and K_cat/K_m values of MpiCP-1 for Z-Tyr-Glu at pH 4.0 and 37°C were 1.33 mM, 1.49 mM min^–1, 723 s^–1 and 545 mM^–1 s^–1, and those of MpiCP-2 at pH 3.5 and 37°C were 1.55 mM, 1.54 mM min^–1, 2,039 s^–1 and 1,318 mM^–1 s^–1, respectively.     

Identification and optimization of tyrosine hydroxylase activity in Mucuna pruriens DC. var. utilis

Tyrosine hydroxylase, an iron containing tetrahydrobiopterin dependent monooxygenase (tyrosine 3-monooxygenase; EC 1.14.16.2), catalyzes the rate-limiting step in which l-dopa is formed from the substrate l-tyrosine. l-Dopa concentration and activity of l-tyrosine hydroxylase enzyme were measured in roots, stem, leaves, pods, and immature seeds of Mucuna pruriens. Immature seeds contained maximum l-dopa content and mature leaves possessed maximum catalytic activity of tyrosine hydroxylase. Tyrosine hydroxylase from leaf homogenate was characterized as a 55 kDa protein by SDS-PAGE and Western-blot analysis with monoclonal mouse IgG2a tyrosine hydroxylase antibody. The conditions for maximum tyrosine hydroxylase activity from the leaf extract were optimized with respect to temperature, pH, cofactor 6-MPH₄, and divalent metal ions. The tyrosine hydroxylase from leaf extract possessed a K _m value of 808.63 μM for l-tyrosine at 37°C and pH 6.0. The activity of the enzyme was slightly inhibited at 2,000 μM l-tyrosine. Higher concentrations of the cofactor 6-MPH₄, however, completely inhibited the synthesis of l-dopa. Tyrosine hydroxylase converted specific monophenols such as l-tyrosine (808.63 μM) and tyramine (K _m 1.1 mM) to diphenols l-dopa and dopamine, respectively. Fe(II) activated the enzyme while higher concentration of other divalent metals reduced its activity. For the first time, tyrosine hydroxylase from M. pruriens is being reported in this study.     

A novel <Emphasis Type="SmallCaps">l</Emphasis>-aspartate dehydrogenase from the mesophilic bacterium <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PAO1: molecular characterization and application for <Emphasis Type="SmallCaps">l</Emphasis>-aspartate production

l-aspartate dehydrogenase (EC 1.4.1.21; l-AspDH) is a rare member of amino acid dehydrogenase superfamily and so far, two thermophilic enzymes have been reported. In our study, an ORF PA3505 encoding for a putative l-AspDH in the mesophilic bacterium Pseudomonas aeruginosa PAO1 was identified, cloned, and overexpressed in Escherichia coli. The homogeneously purified enzyme (PaeAspDH) was a dimeric protein with a molecular mass of about 28 kDa exhibiting a very high specific activity for l-aspartate (l-Asp) and oxaloacetate (OAA) of 127 and 147 U mg⁻¹, respectively. The enzyme was capable of utilizing both nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) as coenzyme. PaeAspDH showed a T _m value of 48°C for 20 min that was improved to approximately 60°C by the addition of 0.4 M NaCl or 30% glycerol. The apparent K _m values for OAA, NADH, and ammonia were 2.12, 0.045, and 10.1 mM, respectively; comparable results were observed with NADPH. The l-Asp production system B consisting of PaeAspDH, Bacillus subtilis malate dehydrogenase and E. coli fumarase, achieved a high level of l-Asp production (625 mM) from fumarate in fed-batch process with a molar conversion yield of 89.4%. Furthermore, the fermentative production system C released 33 mM of l-Asp after 50 h by using succinate as carbon source. This study represented an extensive characterization of the mesophilic AspDH and its potential applicability for efficient and attractive production of l-Asp. Our novel production systems are also hopeful for developing the new processes for other compounds production.     

Purification and Characterization of Carboxypeptidases from the Sarcocarp of Watermelon

Two kinds of carboxypeptidases (F–I, F–II) were purified from the sarcocarp of watermelon (Citrullus vulgaris, var. Shimao). F–I was not purified to homogeneity. F–II was homogeneous on ultracentrifugal analysis, but a trace of impurity was detected at high concentrations by disc electrophoresis.

F–I was optimally active and stable at pH 5.0~5.5 and was strongly inhibited by DFP and HgCl₂, but not by EDTA. The molecular weight and isoelectric point were 89,000 and 4.4, respectively.

F–II was optimally active at pH 5.0 ~ 5.5 and was most stable at pH 5.5 ~ 7.0. It was completely inhibited by DFP and HgCl₂, but not by EDTA and 1, 10-phenanthroline, and it hydrolyzed an oligopeptide containing proline, glutamic acid, lysine and several neutral amino acids, sequentially from the C-terminal. The molecular weight and isolelectric point were 110,000 (5.1 S) and 5.0, respectively.

The similarity of enzymatic properties of both the present enzymes to those of other plant carboxypeptidases and pig kidney cathepsin A are discussed.     

A new alkaline proteinase with pI 2.8 from alkalophilicBacillus sp.

A new serine alkaline proteinase (ALPase II) was purified from the culture broth of an alkalophilicBacillus sp. NKS-21. The molecular weight of ALPase II was estimated to be 32,000 by SDS-polyacrylamide gel electrophoresis. The enzyme had a very low isoelectric point (pI), which was determined to be 2.8. An optimum pH of this enzyme was 10.2. The specific activity was 0.28 katal/kg of protein for milk casein, 0.34 katal/kg for succinyl-l-alanyl-l-alanyl-l-prolyl-l-phenylalanyl-4-methyl-coumaryl-7-amide (Suc-Ala-Ala-Pro-Phe-MCA) and 8.5 katal/kg for succinyl-l-alanyl-l-alanyl-l-prolyl-l-phenylalanyl-p-nitroanilide (Suc-Ala-Ala-Pro-Phe-pNA).The substrate specificity of the alkaline proteinase was studied with the synthetic fluorogenic and chromogenic substrates. It was most favorable for the enzyme that the P₁ site of the substrate might be hydrophobic and bulky amino residue (Phe or Tyr). When the substrate contained four amino residues, the proteinase efficiently expressed its activity. The alkaline proteinase had higher specificity than those of the bacterial serine proteinases, subtilisins Carlsberg and BPN, and lower specificity than that of serine alkaline proteinase with pI 8.2 (ALPase I) obtained from the same bacteria NKS-21. ALPase II did not react with the anti-ALPase I antiserum.     

Mechanism of l-methionine overproduction by Escherichia coli: the replacement of Ser-54 by Asn in the MetJ protein causes the derepression of l-methionine biosynthetic enzymes

We derived l-methionine-analogue-resistant mutants from Escherichia coli JM109 strain by mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine and selected the potent l-methionine-overproducing strains by microbioassay using lactic acid bacteria. One of the mutants, strain TN1, produced approximately 910 mg l-methionine/l following the addition of 0.1% yeast extract to fundamental medium containing glucose and ammonium sulfate. The l-methionine biosynthetic enzymes, cystathionine γ-synthase and cystathionine β-lyase, of the l-methionine-overproducing mutants were little repressed by l-methionine. To analyse the mechanism of l-methionine overproduction in the mutant strains, the metJ gene coding for the E. colimet repressor, MetJ protein, was cloned and sequenced by the polymerase chain reaction. The same single-amino-acid subsitution (wild-type Ser → Asn) at position 54 was observed in four independent l-methionine-producing mutants. When the wild-type metJ gene was then introduced into strain TN1 having the mutant metJ gene, the level of enzyme synthesis and the l-methionine productivity in the transformants were found to revert to those of the wild-type. It was therefore considered that only one point mutation in the metJ gene occurred in the l-methionine-producing mutants. These results demonstrate the important role of residue 54 of the MetJ protein in l-methionine overproduction, probably because of the derepression of l-methionine biosynthetic enzymes. Received: 6 January 1999 / Received last revision: 19 February 1999 / Accepted: 26 February 1999     

Morpholine-induced formation of l-alanine dehydrogenase activity in Mycobacterium strain HE5

An NAD-dependent, morpholine-stimulated l-alanine dehydrogenase activity was detected in crude extracts from morpholine-, pyrrolidine-, and piperidine-grown cells of Mycobacterium strain HE5. Addition of morpholine to the assay mixture resulted in an up to 4.6-fold increase of l-alanine dehydrogenase activity when l-alanine was supplied at suboptimal concentration. l-Alanine dehydrogenase was purified to near homogeneity using a four-step purification procedure. The native enzyme had a molecular mass of 160 kDa and contained one type of subunit with a molecular mass of 41 kDa, indicating a tetrameric structure. The sequence of 30 N-terminal amino acids was determined and showed a similarity of up to 81% to that of various alanine dehydrogenases. The pH optimum for the oxidative deamination of l-alanine, the only amino acid converted by the enzyme, was determined to be pH 10.1, and apparent K _m values for l-alanine and NAD were 1.0 and 0.2 mM, respectively. K _m values of 0.6, 0.02, and 72 mM for pyruvate, NADH, and NH₄ ⁺, respectively, were estimated at pH 8.7 for the reductive amination reaction. Received: 25 September 1998 / Accepted: 11 March 1999     

Amino acid metabolism in the thermophilic phototroph,Chloroflexus aurantiacus: properties and metabolic role of two l-threonine (l-serine) dehydratases

Two l-threonine (l-serine) dehydratases (EC 4.2.1.16) of the thermophilic phototrophic bacterium Chloroflexus aurantiacus Ok-70-fl were purified to electrophoretic homogeneity by procedures involving anion exchange and hydrophobic interaction chromatography. Only one of the two enzymes was sensitive to inhibition by l-isoleucine (K _i=2 M) and activation by l-valine. The isoleucine-insensitive dehydratase was active with l-threonine (K _m=20 mM) as well as with l-serine (K _m=10 mM) whereas the other enzyme, which displayed much higher affinity to l-threonine (K _m=1.3 mM), was inactivated when acting on l-serine. Both dehydratases contained pyridoxal-5-phosphate as cofactor. When assayed by gel filtration techniques at 20 to 25° C, the molecular weights of both enzymes were found to be 106,000±6,000. In sodium dodecylsulfate-polyacrylamide gel electrophoresis, the two dehydratases yielded only one type of subunit with a molecular weight of 55,000±3,000. The isoleucine-insensitive enzyme was subject to a glucose-mediated catabolite repression.Abbreviations A absorbance - ile isoleucine - PLP pyridoxal-5-phosphate - SDS sodium dodecyl sulfate - TDH threonine dehydratase - U unit     

Chromosomal effects on peptidase activities inDrosophila melanogaster

The peptidase system inDrosophila melanogaster (dipeptidase-A, -B, and -C and leucine aminopeptidases G and P) was used as a model to study the effects of modifier genes on activity of enzymes with similar functions. A screen of X, second, and third chromosome substitution isogenic lines revealed the presence of activity modifiers for peptidases on all three chromosomes. Correlation analyses indicated that covariation between some of the peptidase activities is independent of genetic background, while others are associated with variable second chromosomes. Chromosome-specific effects onK _m ,V _max, and specific activity of partially purified peptidases were also detected. Moreover, a repeatable technique using anion-exchange column chromatography allowed the characterization of possibly two putative peptidic enzymes, glycyl-l-isoleucine-ase andl-leucyl-l-proline-ase, whose kinetic properties differ from the dipeptidases and the leucine aminopeptidases. These findings confirm the existence of activity modifiers for peptidases, much like other enzymes inDrosophila melanogaster. These studies were supported by grants from the National Institutes of Health (GM42-115-01A1), the Whitaker Foundation of the Research Corporation (C-2560), and the National Science Foundation (USE 8951018) to Kazuo Hiraizumi.     

Partial purification and properties of γ-glutamyltranspeptidase from mycelia of Morchella esculenta

Three -glutamyltranspeptidase (enzymes I, II and III) were partially purified from the cell free extracts of the cultured mycelia of Morchella esculenta Fr. The molecular masses of enzymes were 155,000 (I), 219,000 (II) and 102,000 (III). All of them catalyzed both hydrolysis and transpeptidation of various -glutamyl compounds. -l-Glutamyl-cis-3-amino-l-proline occurring in the cultured mycelia of this fungus was a good substrate for both reactions. K _m values for hydrolysis were in the order of 10^-4 to 10^-5 M, and those for transpeptidation were in the order of 10^-2 to 10^-4 M. The enzymes were inhibited by a -glutamyltranspeptidase inhibitor, l-serine plus borate.Abbreviations -GTP -glutamyltranspeptidase - HPLC High-performance liquid chromatography     

Characterization of a recombinant endo-1,5-α-<Emphasis Type="SmallCaps">l</Emphasis>-arabinanase from the isolated bacterium <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>

The bacterium Bacillus licheniformis, which exhibits high hydrolytic activity toward arabinan, was isolated from soil, and its gene encoding endo-1,5-α-l-arabinanase was cloned and sequenced. The gene has an open reading frame that encodes 328 amino acids, including a signal peptide of 37 amino acids. Endo-1,5-α-l-arabinanase, a member of glycosyl hydrolase family 43, was expressed in Escherichia coli and purified as a 34-kD monomer with a specific activity of 27 U/mg. Optimal activity toward debranched arabinan (linear 1,5-α-l-arabinan) occurred at pH 6.0 and 35°C, with a k _cat of 160/sec and a K _m of 19 mg/mL.     

Purification and characterization of mouse hepatic enzyme that converts selenomethionine to methylselenol by its α,γ-elimination

The objective of this study was to purify and characterize a mouse hepatic enzyme that directly generates CH₃SeH from seleno-l-methionine (l-SeMet) by the α,γ-elimination reaction. The l-SeMet α,γ-elimination enzyme was ubiquitous in tissues from ICR mice and the activity was relatively high in the large intestine, brain, and muscle, as well as the liver. Aging and sex of the mice did not have any significant influence on the activity in the liver. The enzyme was purified from the mouse liver by ammonium sulfate precipitation and four kinds of column chromatography. These procedures yielded a homogeneous enzyme, which was purified approx 1000-fold relative to mouse liver extract. Overall recovery was approx 8%. The purified enzyme had a molecular mass of approx 160 kDa with four identical subunits. The K _m value of the enzyme for the catalysis of l-SeMet was 15.5 m M, and the V _max was 0.29 units/mg protein. Pyridoxal 5′-phosphate (pyridoxal-P) was required as a cofactor because the holoenzyme could be resolved to the apoenzyme by incubation with hydroxylamine and reconstituted by addition of pyridoxal-P. The enzyme showed the optimum activity at around pH 8.0 and the highest activity at 50°C; it catalyzed the α,γ-elimination reactions of several analogs such as d,l-homocysteine and l-homoserine in addition to l-SeMet. This enzyme also catalyzed the α,β-elimination reaction of Se-methylseleno-l-cysteine. However, l-methionine was inerts. Therefore, the purified enzyme was different from the bacterial l-methionine γ-lyase that metabolizes l-SeMet to CH₃SeH, in terms of the substrate specificity. These results were the first identification of a mammalian enzyme that specifically catalyzes the α,γ-elimination reaction of l-SeMet and immediately converts it to CH₃SeH, an important metabolite of Se.     

Synthesis and evaluation of sulfonamide-bearing thiazole as carbonic anhydrase isoforms hCA I and hCA II

Sulfonamide-bearing thiazole compounds were synthesized and their inhibitory effects on the activity of purified human carbonic anhydrase I and II were evaluated. Human carbonic anhydrase isoenzymes (hCA-I and hCA-II) were purified from erythrocyte cells by affinity chromatography. The inhibitory effects of the 12 synthesized sulfonamide (5a–l) on the hydratase and esterase activities of these isoenzymes (hCA-I and hCA-II) were studied in vitro. In relation to these activities, the inhibition equilibrium constants (Ki) were determined. The results showed that all the synthesized compounds inhibited the CA isoenzyme activity. Among them 5b was found to be the most active (IC_50?=_?0.35?μM; Ki: 0.33?μM) for hCA I and hCA II.     

Recombinant production of serine hydroxymethyl transferase from Streptococcus thermophilus and its preliminary evaluation as a biocatalyst

The glyA gene encoding a serine hydroxymethyl transferase (SHMT) with threonine aldolase activity was isolated from Streptococcus thermophilus YKA-184 chromosomal DNA. This aldolase is a pyridoxal 5′-phosphate-dependent enzyme that stereospecifically catalyzes the interconversion of l-threonine to glycine and acetaldehyde. The enzyme was overexpressed in Escherichia coli M15 as a recombinant protein of 45 kDa with a His6-tag at its N-terminus. The recombinant enzyme was purified to homogeneity by a single chromatographic step using Ni-nitrilotriacetic acid affinity, obtaining a high activity-recovery yield (83%). Lyophilized and precipitated enzymes were stable at least for 10 weeks when stored at −20°C and 4°C. It was observed that the K _m for l-allo-threonine was 38-fold higher than that for l-threonine, suggesting this enzyme can be classified as a specific l-allo-threonine aldolase. The optimum pH range of threonine aldolase activity for the recombinant SHMT was pH 6–7. When tested for aldol addition reactions with non-natural aldehydes, such as benzyloxyacetaldehyde and (R)-N-Cbz-alaninal, two possible β-hydroxy-α-amino acid diastereoisomers were produced, but with moderate stereospecificity. The enzyme showed potential as a biocatalyst for the stereoselective synthesis of β-hydroxy-α-amino acids.