D-threonine Aldolase Research Articles

Structure of pyridoxal 5'-phosphate-bound D-threonine aldolase from Chlamydomonas reinhardtii.

D-Threonine aldolase (DTA) is a pyridoxal-5'-phosphate-dependent enzyme which catalyzes the reversible aldol reaction of glycine with a corresponding aldehyde to yield the D-form β-hydroxy-α-amino acid. This study produced and investigated the crystal structure of DTA from Chlamydomonas reinhardtii (CrDTA) at 1.85 Å resolution. To our knowledge, this is the first report on the crystal structure of eukaryotic DTA. Compared with the structure of bacterial DTA, CrDTA has a similar arrangement of active-site residues. On the other hand, we speculated that some non-conserved residues alter the affinity for substrates and inhibitors. The structure of CrDTA could provide insights into the structural framework for structure-guided protein engineering studies to modify reaction selectivity.

Highly selective kinetic resolution of D/L-syn-p-sulfone phenylserine catalyzed by d-threonine aldolase in two-phase ionic solvent

In the chemical synthesis of L-syn-p-methylsulfoxide phenylserine ethyl ester (D-ethyl ester), l-tartaric acid or enzymatic resolution is employed to resolve the racemate, and thus obtain the target compound, and the remaining isomer can be recycled to obtain the raw material. In this study, high-purity L-syn-p-methylsulfoxide phenylserine (L-syn-MPS) was obtained. The kinetics of the d-threonine aldolase enzymatic hydrolysis reaction reveals that D-syn-p-sulfoxylphenylserine resolves well in [BMIM][BF4] ionic solvents. The D/L-syn-MPS racemate was resolved using a two-phase ionic solvent [BMIM][NTf2] to afford L-syn-MPS (ee (enantiomeric excess) > 99%) and a white solid in 41.7% yield. Therefore, this system is suitable for the separation of insoluble aldehydes and successfully avoids the condensation of hydroxyl aldehydes to form D-anti-MPS.

High-Throughput Screening Method for Directed Evolution and Characterization of Aldol Activity of D-Threonine Aldolase.

A rapid and reliable method for the determination of aldol condensation activity of threonine aldolases (TAs) toward aldehydes and glycine was developed. This 2,4-dinitrophenylhydrazine (DNPH) method has high sensitivity and low background disturbance and can be spectrophotometrically measured for high-throughput screening and characterization of TAs. For 4-methylsulfonyl benzaldehyde (MSB), the maximum absorbance peak was observed at around 485nm. Site-directed saturation mutagenesis libraries of D-threonine aldolase from Alcaligenes xylosoxidans CGMCC 1.4257 (AxDTA) was constructed and screened with this DNPH method for increased aldol activity toward MSB. Two beneficial variants AxDTAD321C and AxDTAN101G were identified. Substrate specificity of AxDTA and variants toward nineteen aldehydes with different substituents was facilely characterized employing this DNPH method. Furthermore, AxDTA variants displayed enhanced catalytic performance and selectivity in aldol reaction. Consequently, our study provides a rapid screening and characterization method for TAs with potential applications in preparation of chiral β-hydroxy-α-amino acids.

D-Serine as a Key Building Block: Enzymatic Process Development and Smart Applications within the Cascade Enzymatic Concept

An efficient enzymatic method catalyzed by an enzyme from the d-threonine aldolase (DTA) family was developed for d-serine production at industrial scale. This process was used for the synthesis of two valuable ketoses, l-erythrulose and d-fructose, within the cascade enzymatic concept involving two other enzymes. Indeed, d-serine was used as a substrate of d-amino acid oxidase (DAAO) for the in situ generation of the corresponding α-keto acid, hydroxypyruvic acid (HPA), a key donor substrate of transketolase (TK). This enzyme catalyzed the irreversible transfer of the ketol group from HPA to an aldehyde acceptor to form a (3S)-ketose by stereoselective carbon–carbon bond formation. The compatibility of all enzymes and substrates allowed a sequential three-step enzymatic process to be performed without purification of the intermediates. This strategy was validated with two TK aldehyde substrates to finally obtain the corresponding (3S)-ketoses with high control of the stereoselectivity and excellent aldehyde conversion rates.

Dynamic Kinetic Resolution for Asymmetric Synthesis of L‐Noncanonical Amino Acids from D‐Ser Using Tryptophan Synthase and Alanine Racemase

L‐Ser is often used to synthesize some significant l‐noncanonical α‐amino acids(l‐ncAAs), which are the prevalent intermediates and precursors for functional synthetic compounds. In this study, threonine aldolase from Escherichia coli k‐12 MG1655 has been used to synthesize l‐Ser. In contrast to the maximum catalytic capacity (20 g/L) for l‐threonine aldolase(LTA), d‐Ser was synthesized with high yield (240 g/L) from cheap Gly and paraformaldehyde using d‐threonine aldolase (DTA) from Arthrobacter sp ATCC. In order to fully utilize d‐Ser and expand the resource of l‐Ser, a dynamic kinetic resolution system was constructed to convert d/dl‐Ser to l‐Ser through combining alanine racemase (Alr) from Bacillus subtilis with l‐tryptophan synthase (TrpS) from Escherichia coli k‐12 MG1655, and l‐ncAAs including l‐Trp and l‐Cys derivatives were synthesized with excellent enantioselectivity and in high yields. The results indicated l‐ncAAs could be efficiently synthesized from d‐Ser using this original and green dynamic kinetic resolution system, and the reliable l‐Ser resource has been established from simple and achiral substrates.

Crystallization and X-ray analysis of D-threonine aldolase from Chlamydomonas reinhardtii.

D-Threonine aldolase from the green alga Chlamydomonas reinhardtii (CrDTA) catalyzes the interconversion of several β-hydroxy-D-amino acids (e.g. D-threonine) and glycine plus the corresponding aldehydes. Recombinant CrDTA was overexpressed in Escherichia coli and purified to homogeneity; it was subsequently crystallized using the hanging-drop vapour-diffusion method at 295 K. Data were collected and processed at 1.85 Å resolution. Analysis of the diffraction pattern showed that the crystal belonged to space group P1, with unit-cell parameters a = 64.79, b = 74.10, c = 89.94 Å, α = 77.07, β = 69.34, γ=71.93°. The asymmetric unit contained four molecules of CrDTA. The Matthews coefficient was calculated to be 2.12 Å3 Da-1 and the solvent content was 41.9%.

Cloning and characterization of d-threonine aldolase from the green alga Chlamydomonas reinhardtii

d-Threonine aldolase (DTA) catalyzes the pyridoxal 5’-phosphate (PLP)-dependent interconversion of d-threonine and glycine plus acetaldehyde. The enzyme is a powerful tool for the stereospecific synthesis of various β-hydroxy amino acids in synthetic organic chemistry. In this study, DTA from the green alga Chlamydomonas reinhardtii was discovered and characterized, representing the first report to describe the existence of eukaryotic DTA. DTA was overexpressed in recombinant Escherichia coli BL21 (DE3) cells; the specific activity of the enzyme in the cell-free extract was 0.8 U/mg. The recombinant enzyme was purified to homogeneity by ammonium sulfate fractionation, DEAE-Sepharose, and Mono Q column chromatographies (purified enzyme 7.0 U/mg). For the cleavage reaction, the optimal temperature and pH were 70 °C and pH 8.4, respectively. The enzyme demonstrated 90% of residual activity at 50 °C for 1 h. The enzyme catalyzed the synthesis of d- and d-allo threonine from a mixture of glycine and acetaldehyde (the diastereomer excess of d-threonine was 18%). DTA was activated by several divalent metal ions, including manganese, and was inhibited by PLP enzyme inhibitors and metalloenzyme inhibitors.

Synthesis of a Chiral β-Hydroxy-α-amino Acid by Using d-Threonine Aldolase

Synthesis of a Chiral β-Hydroxy-α-amino Acid by Using d-Threonine Aldolase

Preparation of β-hydroxy-α-amino Acid Using Recombinant d-Threonine Aldolase

The chiral β-hydroxy-α-amino acid, (2R,3S)-2-amino-3-hydroxy-3-(pyridin-4-yl)-propanoic acid, is a key intermediate in the synthesis of the API (2R,3S)-2-amino-3-hydroxy-3-(pyridin-4-yl)-1-(pyrrolidin-1-yl)propan-1-one, a developmental drug candidate. Two d-threonine aldolase enzymes were identified to catalyze the aldol addition of glycine and pyridine 4-carboxaldehyde for the synthesis of the β-hydroxy-α-amino acid. The two d-threonine aldolase enzymes have similar properties. Efficient recombinant E. coli fermentation processes were developed for producing the enzymes. The stabilities of the enzymes were significantly improved by addition of divalent cations. An unexpected and beneficial finding was that the β-hydroxy-α-amino acid aldol addition product directly crystallized out from the reaction mixture in high purity and high diastereo- and enantioselectivity, contributing also to high yield and allowing easy isolation, processing, and downstream utilization. The temperature, pH, and amounts of reactants and enzyme were optimized to minimize reaction time and enzyme and raw material usage and maximize amino acid formation. Efficient d-threonine aldolase-catalyzed synthesis and recovery of the β-hydroxy-α-amino acid at the 100 L scale was demonstrated leading to a highly efficient and environmentally friendly process for the production of the API.

D-Serine Dehydratase from Chicken Kidney: A Vertebral Homologue of the Cryptic Enzyme from Burkholderia cepacia

D-serine dehydratase (DSD) catalyses the conversion of d-serine to pyruvate and ammonia. d-Serine is a physiological modulator of glutamate neurotransmission in vertebrate brains. In mammals d-serine is degraded by d-amino-acid oxidase, whereas in chicken brain it is degraded by DSD, as we have recently demonstrated [Tanaka et al. (2007) Anal. Biochem. 362, 83-88]. To clarify the roles of DSD in avian species, we purified DSD from chicken kidney. The purified enzyme was a heterodimer consisting of subunits separable by SDS-PAGE but with identical N-terminal amino acid sequences. The prominent absorption at 416 nm and the inhibition of the enzyme both by hydroxylamine and by aminooxyacetate suggested that the enzyme contains pyridoxal 5'-phosphate as a cofactor. The enzyme showed the highest specificity to d-serine: the k(cat)/K(m) values of DSD for d-serine, d-threonine and l-serine were 6.19 x 10(3), 164 and 16 M(-1)s(-1), respectively. DSD was found immunohistochemically in the proximal tubules of the chicken kidney. Judging from the amino acid sequence deduced from the cDNA, chicken DSD is a homologue of cryptic DSD from Burkholderia cepacia and low-specificity d-threonine aldolase from Arthrobacter sp. strain DK-38, all of which have a cofactor binding motif of PHXK(T/A) in their N-terminal portions.

Synthesis of γ-halogenated and long-chain β-hydroxy-α-amino acids and 2-amino-1,3-diols using threonine aldolases

The l- and d-threonine aldolase catalyzed formation of γ-halogenated and long-chain l- and d-3-alkylserine-derivatives 1– 12, respectively, was shown starting from glycine and halogenated C 2– or C 4–C 12 aldehydes. lTA from Pseudomonas putida accepted all tested aldehydes with strongly varying diastereoselectivity (de up to 93%). Only aldehydes smaller than decanal were converted by dTA from Alcaligenes xylosoxidans with good selectivities (de up to 73%). Utilizing isobutanal enantio- and diastereopure d- syn-2-amino-3-hydroxy-4-methylpentanoic acid was obtained (ee>99%, de>95%), which was converted to the corresponding 2-amino-1,3-diol.

Threonine aldolases—an emerging tool for organic synthesis

In a systematic study, 21 ring-substituted benzaldehydes were reacted with glycine under catalysis with a l-threonine aldolase ( lTA) from Pseudomonas putida and a d-threonine aldolase ( dTA) from Alcaligenes xylosoxidans to form the corresponding β-hydroxy-α-amino acids 1– 18. dTA proved to be highly selective with ee's >99% ( d) and de's up to 99% ( syn). Two thiamphenicol precursors were synthesized utilizing dTA on a preparative scale. lTA-catalyzed reactions led to ee's >99% ( l) but low to moderate de's (20–50%, syn).

A novel enzyme, D-3-hydroxyaspartate aldolase from Paracoccus denitrificans IFO 13301: purification, characterization, and gene cloning.

A novel enzyme, D-3-hydroxyaspartate aldolase (D-HAA), catalyzing the conversion of D-3-hydroxyaspartate to glyoxylate plus glycine, was purified to homogeneity from Paracoccus denitrificans IFO 13301. D-HAA is strictly D-specific as to the alpha-position, whereas the enzyme does not distinguish between threo and erythro forms at the beta-position. In addition to D-3-hydroxyaspartate, the enzyme also acts on d-threonine, D-3-3,4-dihydroxyphenylserine, D-3-3,4-methylenedioxyphenylserine, and D-3-phenylserine. The D-HAA gene was cloned and sequenced. The gene contains an open reading frame consisting of 1,161 nucleotides corresponding to 387 amino acid residues. The predicted amino acid sequence displayed 35% and 22% identity with that of the D-threonine aldolase of Arthrobacter sp. DK-38 and Alcaligenes xylosoxidan IFO 12669, respectively. This is the first paper reporting both a purified enzyme with D-3-hydroxyaspartate aldolase activity and also its gene cloning.

Gene cloning and overproduction of low-specificity D-threonine aldolase from Alcaligenes xylosoxidans and its application for production of a key intermediate for parkinsonism drug.

The dtaAX gene encoding a pyridoxal 5'-phosphate (pyridoxal-P)-dependent low-specificity D-threonine aldolase was cloned from the chromosomal DNA of Alcaligenes xylosoxidans IFO 12669. It contains an open reading frame consisting of 1,134 nucleotides corresponding to 377 amino acid residues. The predicted amino acid sequence displayed 54% identity with that of D-threonine aldolase from gram-positive bacteria Arthrobacter sp. DK-38, but showed no significant similarity with those of other known pyridoxal-P enzymes. This gram-negative bacterial enzyme was highly overproduced in recombinant Escherichia coli cells, and the specific activity of the enzyme in the cell extract was as high as 18 U/mg (purified enzyme 38.6 U/mg), which was 6,000 times higher than that from the wild-type Alcaligenes cell extract. The recombinant enzyme was thus feasibly purified to homogeneity by ammonium sulfate fractionation and DEAE-Toyopearl chromatography steps. The recombinant low-specificity D-threonine aldolase was shown to be an efficient biocatalyst for resolution of L-beta-3,4-methylenedioxyphenylserine, an intermediate for production of a therapeutic drug for Parkinson's disease.

A new route to L-threo-3-[4-(methylthio)phenylserine], a key intermediate for the synthesis of antibiotics: recombinant low-specificity D-threonine aldolase-catalyzed stereospecific resolution.

A new enzymatic resolution process was established for the production of L-threo-3-[4-(methylthio)phenylserine] (MTPS), an intermediate for synthesis of antibiotics, florfenicol and thiamphenicol, using the recombinant low-specificity D-threonine aldolase from Arthrobacter sp. DK-38. Chemically synthesized DL-threo-MTPS was efficiently resolved with either the purified enzyme or the intact recombinant Escherichia coli cells overproducing the enzyme. Under the optimized experimental conditions, 100 mM (22.8 g l-1) L-threo-MTPS was obtained from 200 mM (45.5 g l-1) DL-threo-MTPS, with a molar yield of 50% and a 99.6% enantiomeric excess.

A Novel Metal-activated Pyridoxal Enzyme with a Unique Primary Structure, Low Specificity d-Threonine Aldolase fromArthrobacter sp. Strain DK-38: MOLECULAR CLONING AND COFACTOR CHARACTERIZATION

The gene encoding low specificity D-threonine aldolase, catalyzing the interconversion of D-threonine/D-allo-threonine and glycine plus acetaldehyde, was cloned from the chromosomal DNA of Arthrobacter sp. strain DK-38. The gene contains an open reading frame consisting of 1,140 nucleotides corresponding to 379 amino acid residues. The enzyme was overproduced in recombinant Escherichia coli cells and purified to homogeneity by ammonium sulfate fractionation and three-column chromatography steps. The recombinant aldolase was identified as a pyridoxal enzyme with the capacity of binding 1 mol of pyridoxal 5'-phosphate per mol of subunit, and Lys59 of the enzyme was determined to be the cofactor binding site by chemical modification with NaBH4. In addition, Mn2+ ion was demonstrated to be an activator of the enzyme, although the purified enzyme contained no detectable metal ions. Equilibrium dialysis and atomic absorption studies revealed that the recombinant enzyme could bind 1 mol of Mn2+ ion per mol of subunit. Remarkably, the predicted amino acid sequence of the enzyme showed no significant similarity to those of the currently known pyridoxal 5'-phosphate-dependent enzymes, indicating that low specificity D-threonine aldolase is a new pyridoxal enzyme with a unique primary structure. Taken together, low specificity D-threonine aldolase from Arthrobacter sp. strain DK-38, with a unique primary structure, is a novel metal-activated pyridoxal enzyme.

Enzymatic Synthesis of β-Hydroxy-α-amino Acids Based on Recombinant d- and l-Threonine Aldolases

To exploit the enzymatic method for the synthesis of β-hydroxy-α-amino acids, the genes coding for the Escherichia coli l-threonine aldolase (LTA; EC 2.1.2.1) and Xanthomonus oryzae d-threonine aldolase (DTA) were cloned and overexpressed in E. coli through primer-directed polymerase chain reactions. The purified recombinant enzymes were studied with respect to kinetics, specificity, stability, additive requirement, temperature profile, and pH dependency. DTA requires magnesium ion as a cofactor, while LTA needs no metal ions. These enzymes work well in the presence of DMSO with concentration up to 40%, and DMSO-induced rate acceleration of LTA-catalyzed reaction was observed. Both enzymes use pyridoxal phosphate coenzyme to activate glycine to react with a wide range of aldehydes. LTA gave erythro-β-hydroxy-α-l-amino acids with aliphatic aldehydes and the threo isomer with aromatic aldehydes as kinetically controlled products. On the other hand, DTA formed threo-β-hydroxy-α-d-amino acids as kinetically controlled products with aliphatic and aromatic aldehydes but the diastereoselectivity was lower than that of LTA. Under optimal conditions, several β-hydroxy-α-amino acid derivatives (3-hydroxyleucines, γ-benzyloxythreonines, γ-benzyloxymethylthreonines, and polyoxamic acids) have been stereoselectively synthesized on preparative scales using these enzymes. Also, the tandem use of DTA and phosphatases has made possible the synthesis and separation of d-allo-threonine phosphate and d-threonine.

Isolation and characterization of D-threonine aldolase, a pyridoxal-5'-phosphate-dependent enzyme from Arthrobacter sp. DK-38.

D-Threonine aldolase is an enzyme that catalyzes the cleavage of D-threonine into glycine and acetaldehyde. Its activity was found in several genera of bacteria such as Arthrobacter, Alcaligenes, Xanthomonas, and Pseudomonas, but not in yeasts or fungi. The enzyme was purified to homogeneity from one strain, Arthrobacter sp. DK-38. The enzyme appeared to consist of a single polypeptide chain with an apparent molecular mass of 51 kDa. This enzyme, as well as L-threonine aldolase, requires pyridoxal 5'-phosphate (pyridoxal-P) as a coenzyme. Unlike other pyridoxal-P enzymes, D-threonine aldolase also requires a divalent cation such as Co2+, Ni2+, Mn2+, or Mg2+ for its catalytic activity. The enzyme completely lost its activity in the absence of either pyridoxal-P or a divalent cation. A divalent cation was also essential for the thermal stability of the enzyme. The metal-free enzyme tends to become thermally unstable, resulting in the irreversible loss of its catalytic activity. The enzyme is strictly D-specific for the alpha-position, whereas it cannot distinguish between threo and erythro forms at the beta-position. Thus, D-threonine and D-allothreonine act as substrates of the enzyme, but their kinetic parameters are different; the Km and Vmax values are 3.81 mM and 38.8 micromol x min(-1) x mg(-1) toward D-threonine, and 14.0 mM and 102 micromol x min(-1) x mg(-1) toward D-allothreonine. respectively. The aldolase reaction is reversible, and the enzyme is therefore able to produce nearly equimolar amounts of D-threonine and D-allothreonine through C-C bond formation between glycine and acetaldehyde. The enzyme also acts, in the same manner, on several other D-beta-hydroxy-alpha-amino acids, including D-beta-phenylserine, D-beta-hydroxy-alpha-aminovaleric acid, D-beta-3,4-dihydroxyphenylserine, and D-beta-3,4-methylenedioxyphenylserine.