Publications

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

  Kataoka M., Ikemi M., Morikawa T., Miyoshi T., Nishi K., Wada M., Yamada H., Shimizu S.

  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 ... >> More

  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. << Less

  Eur. J. Biochem. 248:385-393(1997) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Threonine aldolase and alanine racemase: novel examples of convergent evolution in the superfamily of vitamin B6-dependent enzymes.

  Paiardini A., Contestabile R., D'Aguanno S., Pascarella S., Bossa F.

  Vitamin B(6)-dependent enzymes may be grouped into five evolutionarily unrelated families, each having a different fold. Within fold type I enzymes, L-threonine aldolase (L-TA) and fungal alanine racemase (AlaRac) belong to a subgroup of structurally and mechanistically closely related proteins, w ... >> More

  Vitamin B(6)-dependent enzymes may be grouped into five evolutionarily unrelated families, each having a different fold. Within fold type I enzymes, L-threonine aldolase (L-TA) and fungal alanine racemase (AlaRac) belong to a subgroup of structurally and mechanistically closely related proteins, which specialised during evolution to perform different functions. In a previous study, a comparison of the catalytic properties and active site structures of these enzymes suggested that they have a catalytic apparatus with the same basic features. Recently, recombinant D-threonine aldolases (D-TAs) from two bacterial organisms have been characterised, their predicted amino acid sequences showing no significant similarities to any of the known B(6) enzymes. In the present work, a comparative structural analysis suggests that D-TA has an alpha/beta barrel fold and therefore is a fold type III B(6) enzyme, as eukaryotic ornithine decarboxylase (ODC) and bacterial AlaRac. The presence of both TA and AlaRac in two distinct evolutionary unrelated families represents a novel and interesting example of convergent evolution. The independent emergence of the same catalytic properties in families characterised by completely different folds may have not been determined by chance, but by the similar structural features required to catalyse pyridoxal phosphate-dependent aldolase and racemase reactions. << Less

  Biochim Biophys Acta 1647:214-219(2003) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• 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.

  Liu J.Q., Odani M., Yasuoka T., Dairi T., Itoh N., Kataoka M., Shimizu S., Yamada H.

  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 p ... >> More

  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. << Less

  Appl Microbiol Biotechnol 54:44-51(2000) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• 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.

  Liu J.Q., Odani M., Dairi T., Itoh N., Shimizu S., Yamada H.

  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 sy ... >> More

  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. << Less

  Appl Microbiol Biotechnol 51:586-591(1999) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• A novel metal-activated pyridoxal enzyme with a unique primary structure, low specificity D-threonine aldolase from Arthrobacter sp. Strain DK-38. Molecular cloning and cofactor characterization.

  Liu J.-Q., Dairi T., Itoh N., Kataoka M., Shimizu S., Yamada H.

  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 corres ... >> More

  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. << Less

  J. Biol. Chem. 273:16678-16685(1998) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.