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Applied and Environmental Microbiology, April 2006, p. 2707-2720, Vol. 72, No. 4
0099-2240/06/$08.00+0 doi:10.1128/AEM.72.4.2707-2720.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Pseudomonad Cyclopentadecanone Monooxygenase Displaying an Uncommon Spectrum of Baeyer-Villiger Oxidations of Cyclic Ketones
Hiroaki Iwaki,1
Shaozhao Wang,2
Stephan Grosse,2
Hélène Bergeron,2
Ayako Nagahashi,1
Jittiwud Lertvorachon,2
Jianzhong Yang,2
Yasuo Konishi,2
Yoshie Hasegawa,1 and
Peter C. K. Lau2*
Department of Biotechnology, Faculty of Engineering and High Technology Research Center, Kansai University, Suita, Osaka 564-8680, Japan,1 Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec H4P 2R2, Canada2
Received 25 November 2005/ Accepted 2 February 2006
Baeyer-Villiger monooxygenases (BVMOs) are biocatalysts that offer the prospect of high chemo-, regio-, and enantioselectivity in the organic synthesis of lactones or esters from a variety of ketones. In this study, we have cloned, sequenced, and overexpressed in Escherichia coli a new BVMO, cyclopentadecanone monooxygenase (CpdB or CPDMO), originally derived from Pseudomonas sp. strain HI-70. The 601-residue primary structure of CpdB revealed only 29% to 50% sequence identity to those of known BVMOs. A new sequence motif, characterized by a cluster of charged residues, was identified in a subset of BVMO sequences that contain an N-terminal extension of 
60 to 147 amino acids. The 64-kDa CPDMO enzyme was purified to apparent homogeneity, providing a specific activity of 3.94 µmol/min/mg protein and a 20% yield. CPDMO is monomeric and NADPH dependent and contains 1 mol flavin adenine dinucleotide per mole of protein. A deletion mutant suggested the importance of the N-terminal 54 amino acids to CPDMO activity. In addition, a Ser261Ala substitution in a Rossmann fold motif resulted in an improved stability and increased affinity of the enzyme towards NADPH compared to the wild-type enzyme (Km = 8 µM versus Km = 24 µM). Substrate profiling indicated that CPDMO is unusual among known BVMOs in being able to accommodate and oxidize both large and small ring substrates that include C11 to C15 ketones, methyl-substituted C5 and C6 ketones, and bicyclic ketones, such as decalone and ß-tetralone. CPDMO has the highest affinity (Km = 5.8 µM) and the highest catalytic efficiency (kcat/Km ratio of 7.2 x 105 M–1 s–1) toward cyclopentadecanone, hence the Cpd designation. A number of whole-cell biotransformations were carried out, and as a result, CPDMO was found to have an excellent enantioselectivity (E > 200) as well as 99% S-selectivity toward 2-methylcyclohexanone for the production of 7-methyl-2-oxepanone, a potentially valuable chiral building block. Although showing a modest selectivity (E = 5.8), macrolactone formation of 15-hexadecanolide from the kinetic resolution of 2-methylcyclopentadecanone using CPDMO was also demonstrated.
* Corresponding author. Mailing address: National Research Council Canada, Biotechnology Research Institute, 6100 Royalmount Avenue, Montreal, Quebec H4P 2R2, Canada. Phone: (514) 496-6325. Fax: (514) 496-6265. E-mail: peter.lau{at}cnrc-nrc.gc.ca.
This publication is issued as NRCC number 47265.
Applied and Environmental Microbiology, April 2006, p. 2707-2720, Vol. 72, No. 4
0099-2240/06/$08.00+0 doi:10.1128/AEM.72.4.2707-2720.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
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