Gene Cloning and Characterization of Two NADH-Dependent 3-Quinuclidinone Reductases from Microbacterium luteolum JCM 9174

Abstract

We used the resting-cell reaction to screen approximately 200 microorganisms for biocatalysts which reduce 3-quinuclidinone to optically pure (R)-(؊)-3-quinuclidinol. Microbacterium luteolum JCM 9174 was selected as the most suitable organism. The genes encoding the protein products that reduced 3-quinuclidinone were isolated from M. luteolum JCM 9174. The bacC gene, which consists of 768 nucleotides corresponding to 255 amino acid residues and is a constituent of the bacilysin synthetic gene cluster, was amplified by PCR based on homology to known genes. The qnr gene consisted of 759 nucleotides corresponding to 252 amino acid residues. Both enzymes belong to the short-chain alcohol dehydrogenase/reductase (SDR) family. The genes were expressed in Escherichia coli as proteins which were His tagged at the N terminus, and the recombinant enzymes were purified and characterized. Both enzymes showed narrow substrate specificity and high stereoselectivity for the reduction of 3-quinuclidinone to (R)-(؊)-3-quinuclidinol. Many methods have been used for the synthesis of optically pure isomers, including enantioselective synthesis, derivatization of natural compounds, and the optical resolution of racemic compounds. Enantioselective organic synthesis is the most efficient and attractive method for producing chiral starting materials for pharmaceuticals and agrochemicals. Chiral metal catalysts such as BINAP-Ru (1) and chiral Co(II) salen complex (2, 3) have been successfully used in producing chiral alcohols or chiral diols from various ketones or epoxides. However, these catalysts are costly and leave trace metal contamination in the products, and they are sometimes difficult to handle. The enzymatic reduction of ketones to optically pure alcohols is more environmentally sustainable and thus more attractive for pharmaceutical manufacturing (4). Several excellent methods using biocatalysts have been reported for producing chiral alcohols (5-10).(R)-(Ϫ)-3-Quinuclidinol, which has a double-ring structure containing nitrogen, is a valuable intermediate for pharmaceuticals. It has been used as the chiral synthone for a cognition enhancer, a bronchodilator, and a urinary incontinence agent (11). The following enzymes have been reported to catalyze the reduction of 3-quinuclidinone to (R)-(Ϫ)-3-quinuclidinol: tropinone reductase, from the plant henbane (Solanaceae, Hyoscyamus niger) (12), and NADPH-dependent 3-quinuclidinone reductase (QNR), from the yeast Rhodotorula rubra (mucilaginosa) (13). Yamamoto et al. reported the production of (R)-(Ϫ)-3-quinuclidinol using recombinant formate dehydrogenase (FDH) and tropinone reductase (14). Uzura et al. reported a reaction system using recombinant glucose dehydrogenase and 3-quinuclidinone reductase from Rhodotorula rubra (13). The optical resolution of (Ϯ)-3-quinuclidinol esters by the hydrolysis reaction of Aspergillus melleus protease was reported by Nomoto et al. (15).In this study, we report two novel 3-quinuclidinone reductase genes, qnr and bacC, from Microbacterium luteolu...

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