title: Purification and gene cloning of an enantioselective thioesterification enzyme from Brevibacterium ketoglutamicum KU1073, a deracemization bacterium of 2-(4-chlorophenoxy)propanoic acid.
abstract: We succeeded in the purification and gene cloning of a new enzyme, α-methyl carboxylic acid deracemizing enzyme 1 (MCAD1) from Brevibacterium ketoglutamicum KU1073, which catalyzes the (S)-enantioselective thioesterification reaction of 2-(4-chlorophenoxy)propanoic acid (CPPA). The cloned gene of MCAD1 contained an ORF of 1,623 bp, encoding a polypeptide of 540 amino acids. In combination with cofactors ATP, coenzyme A (CoASH), and Mg(2+), MCAD1 demonstrated perfect enantioselectivity toward CPPA. The optimal pH and temperature for reaction were found to be 7.25 and 30 °C. Under these conditions, the K(m) and k(cat) values for (S)-CPPA were 0.92 ± 0.17 mM and 0.28 ± 0.026 s(-1) respectively. The results for substrate specificity revealed that MCAD1 had highest activity toward fatty acid tails with a medium chain-length (C(8)). This result indicates that MCAD1 should be classified into a family of medium-chain acyl-CoA synthetase. This novel activity has never been reported for this family.
authors.affiliation: Graduate School of Engineering, University of Hyogo, Himeji, Hyogo, Japan. kato@eng.u-hyogo.ac.jp
authors.forename: Dai-ichiro
authors.initials: D
authors.lastname: Kato
authors.forename: Hiromitsu
authors.initials: H
authors.lastname: Yoshida
authors.forename: Masahiro
authors.initials: M
authors.lastname: Takeo
authors.forename: Seiji
authors.initials: S
authors.lastname: Negoro
authors.forename: Hiromichi
authors.initials: H
authors.lastname: Ohta
mesh_terms: D008456:2-Methyl-4-chlorophenoxyacetic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D001951:Brevibacterium / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D002474:Cell-Free System / Q000201:enzymology; D003001:Cloning, Molecular; D003066:Coenzyme A Ligases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D004795:Enzyme Stability; D004951:Esterification; D008969:Molecular Sequence Data; D010666:Phenylpropionates / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D010666:Phenylpropionates; D011994:Recombinant Proteins; C048213:2-(4-chlorophenoxy)propionic acid; D008456:2-Methyl-4-chlorophenoxyacetic Acid; D003066:Coenzyme A Ligases