RRC ID 3270
Author Iwaki H, Wang S, Grosse S, Bergeron H, Nagahashi A, Lertvorachon J, Yang J, Konishi Y, Hasegawa Y, Lau PC.
Title Pseudomonad cyclopentadecanone monooxygenase displaying an uncommon spectrum of Baeyer-Villiger oxidations of cyclic ketones.
Journal Appl Environ Microbiol
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.
Volume 72(4)
Pages 2707-20
Published 2006-4-1
DOI 10.1128/AEM.72.4.2707-2720.2006
PII 72/4/2707
PMID 16597975
PMC PMC1449013
MeSH Amino Acid Sequence Cloning, Molecular DNA, Bacterial / analysis DNA, Ribosomal / analysis Fatty Acids / chemistry Fatty Acids / metabolism* Hydrocarbons, Alicyclic / metabolism Ketones / metabolism* Mixed Function Oxygenases* / chemistry Mixed Function Oxygenases* / genetics Mixed Function Oxygenases* / metabolism Molecular Sequence Data Oxidation-Reduction Pseudomonas / classification Pseudomonas / enzymology* Pseudomonas / genetics Pseudomonas / growth & development RNA, Ribosomal, 16S / genetics Sequence Analysis, DNA Soil Microbiology Substrate Specificity
IF 4.077
Times Cited 70
Prokaryotes E. coli CV-25(DH5alpha) CV-61(S17-1) ME9040(XL1-Blue)