title: Cloning and characterization of a novel fold-type I branched-chain amino acid aminotransferase from the hyperthermophilic archaeon Thermococcus sp. CKU-1.
abstract: We successfully cloned a novel branched-chain amino acid aminotransferase (Ts-BcAT; EC 2.6.1.42) gene from the Thermococcus sp. CKU-1 genome and expressed it in the soluble fraction of Escherichia coli Rosetta (DE3) cells. Ts-BcAT is a homodimer with an apparent molecular mass of approximately 92 kDa. The primary structure of Ts-BcAT showed high homology with the fold-type I, subgroup I aminotransferases, but showed little homology with BcATs known to date, i.e., those of Escherichia coli and Salmonella typhimurium, which belong to the fold-type IV, subgroup III aminotransferases. The maximum enzyme activity of Ts-BcAT was detected at 95 °C, and Ts-BcAT did not lose any enzyme activity, even after incubation at 90 °C for 5 h. Ts-BcAT was active in the pH range from 4.0 to 11.0, the optimum pH was 9.5, and the enzyme was stable between pH 6 and 7. The exceptionally low pK a of the nitrogen atom in the Lys258 ε-amino group in the internal aldimine bond of Ts-BcAT was determined to be 5.52 ± 0.05. Ts-BcAT used 21 natural and unnatural amino acids as a substrate in the overall transamination reaction. L-Leucine and other aliphatic amino acids are efficient substrates, while polar amino acids except glutamate were weak substrates. Phylogenetic analysis revealed that Ts-BcAT is a novel fold-type I, subgroup I branched-chain aminotransferase.
authors.affiliation: Department of Life Science and Biotechnology, Faculty of Chemistry, Materials and Bioengineering, Kansai University, 3-3-35 Yamate-Cho, Suita, Osaka-Fu, 564-8680, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Uchida
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Hayashi
authors.forename: Tsubasa
authors.initials: T
authors.lastname: Washio
authors.forename: Ryo
authors.initials: R
authors.lastname: Yamasaki
authors.forename: Shiro
authors.initials: S
authors.lastname: Kato
authors.forename: Tadao
authors.initials: T
authors.lastname: Oikawa
mesh_terms: D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001219:Aspartate Aminotransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D008969:Molecular Sequence Data; D019712:Thermococcus / Q000201:enzymology* / Q000235:genetics
chemical_list: D019843:Archaeal Proteins; D001219:Aspartate Aminotransferases