title: Cloning, sequence analysis, and expression in Escherichia coli of the gene encoding monovalent cation-activated levodione reductase from Corynebacterium aquaticum M-13.
abstract: The gene encoding (6R)-2,2,6-trimethyl-1,4-cyclohexanedione (levodione) reductase was cloned from the genomic DNA of the soil isolate bacterium Corynebacterium aquaticum M-13. The gene contained an open reading frame consisting of 801 nucleotides corresponding to 267 amino acid residues. The deduced amino acid sequence showed approximately 35% identity with other short chain alcohol dehydrogenase/reductase (SDR) superfamily enzymes. The probable NADH-binding site and three catalytic residues (Ser-Tyr-Lys) were conserved. The enzyme was sufficiently produced in recombinant Escherichia coli cells using an expression vector pKK223-3, and purified to homogeneity by two-column chromatography steps. The enzyme purified from E. coli catalyzed stereo- and regio-selective reduction of levodione, and was strongly activated by monovalent cations, such as K+, Na+, and NH4+, as was the case of that from C. aquaticum M-13. To our knowledge, this is the first sequencing report of a monovalent cation-activated SDR enzyme.
authors.affiliation: Department of Bioscience, Fukui Prefectural University, Japan.
authors.forename: A
authors.initials: A
authors.lastname: Yoshisumi
authors.forename: M
authors.initials: M
authors.lastname: Wada
authors.forename: H
authors.initials: H
authors.lastname: Takagi
authors.forename: S
authors.initials: S
authors.lastname: Shimizu
authors.forename: S
authors.initials: S
authors.lastname: Nakamori
mesh_terms: D000595:Amino Acid Sequence; D002384:Catalysis; D002412:Cations / Q000494:pharmacology; D003001:Cloning, Molecular; D003352:Corynebacterium / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015723:Gene Library; D005798:Genes, Bacterial / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D002412:Cations; D011994:Recombinant Proteins; D010088:Oxidoreductases; C437407:levodione reductase