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Cloning, Sequence Analysis, and Expression in Escherichia coli of the Gene Encoding Monovalent Cation-activated Levodione Reductase from Corynebacterium aquaticum M-13

2001; Oxford University Press; Volume: 65; Issue: 4 Linguagem: Inglês

10.1271/bbb.65.830

1347-6947

Ayumi YOSHISUMI, Masaru Wada, Hiroshi Takagi, Sakayu Shimizu, Shigeru Nakamori,

Analytical Chemistry and Chromatography

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.