title: Pseudomonad cyclopentadecanone monooxygenase displaying an uncommon spectrum of Baeyer-Villiger oxidations of cyclic ketones.
abstract: 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 approximately 60 to 147 amino acids. The 64-kDa CPDMO enzyme was purified to apparent homogeneity, providing a specific activity of 3.94 micromol/min/mg protein and a 20% yield. CPDMO is monomeric and NADPH dependent and contains approximately 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 (K(m) = 8 microM versus K(m) = 24 microM). 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 C(11) to C(15) ketones, methyl-substituted C(5) and C(6) ketones, and bicyclic ketones, such as decalone and beta-tetralone. CPDMO has the highest affinity (K(m) = 5.8 microM) and the highest catalytic efficiency (k(cat)/K(m) ratio of 7.2 x 10(5) 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.
authors.affiliation: National Research Council Canada, Biotechnology Research Institute, 6100 Royalmount Avenue, Montreal, Quebec H4P 2R2, Canada.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Iwaki
authors.forename: Shaozhao
authors.initials: S
authors.lastname: Wang
authors.forename: Stephan
authors.initials: S
authors.lastname: Grosse
authors.forename: Hélène
authors.initials: H
authors.lastname: Bergeron
authors.forename: Ayako
authors.initials: A
authors.lastname: Nagahashi
authors.forename: Jittiwud
authors.initials: J
authors.lastname: Lertvorachon
authors.forename: Jianzhong
authors.initials: J
authors.lastname: Yang
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Konishi
authors.forename: Yoshie
authors.initials: Y
authors.lastname: Hasegawa
authors.forename: Peter C K
authors.initials: PC
authors.lastname: Lau
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000032:analysis; D004275:DNA, Ribosomal / Q000032:analysis; D005227:Fatty Acids / Q000737:chemistry / Q000378:metabolism*; D006840:Hydrocarbons, Alicyclic / Q000378:metabolism; D007659:Ketones / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011549:Pseudomonas / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA; D012988:Soil Microbiology; D013379:Substrate Specificity
chemical_list: D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D005227:Fatty Acids; D006840:Hydrocarbons, Alicyclic; D007659:Ketones; D012336:RNA, Ribosomal, 16S; C117025:pentadecanoic acid; D006899:Mixed Function Oxygenases