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.

FIG. 1.

Localization of cpdB encoding cyclopentadecanone monooxygenase in between cpdR, which encodes a potential transcriptional regulator, and cpdC, which encodes a lactone hydrolase, within a 4.216-kb BclI chromosomal fragment of Pseudomonas sp. strain HI-70. The XhoI restriction site within the cpdB gene is the point of insertion of a lacZ-Km^r cassette resulting in a mutant strain designated HI-70MB. The numbers in parentheses refer to the nucleotide positions of the respective endonuclease cleavage sites.

FIG. 2.

(A) Identification of a conserved cluster of charged amino acids in the N-terminal sequences of a subset of BVMO sequences. The highly conserved sequences are shaded. CHMO, CPMO, and PAMO are representative BVMOs that do not have the extraneous N-terminal extension. The FAD-binding motif (GxGxxG, in dark shade) serves as a point of reference. In HAPMO, the first 19 amino acids are not shown; an alternative alignment provided a better FAD-binding motif (25). The origins of the various sequences, besides CpdB (this study), are shown as superscripts in the far left column as follows: 1, B. japonicum (Q89NI1); 2, S. avermitilis (Q82IY8); 3, Streptomyces coelicolor (Q9RL17); 4, M. paratuberculosis (Q73U59); 5, R. ruber (Q938F6); 6, P. fluorescens ACB (AAK54073); 7, T. fusca (1W4X_A); 8, Comamonas sp. strain NCIMB 9872 (Q8GAW0); and 9, Acinetobacter sp. strain NCIMB 9871 (BAA86293). The characters in parentheses are GenBank accession or reference numbers. Put., putative; Hyp. Prot., hypothetical protein. (B) Sequence alignment showing an apparent insertional sequence in CpdB and related sequences in between the BVMO fingerprint and the second Rossmann fold motif, as indicated. Amino acids 240 to 246 in CpdB are the deleted region, and the underlined G and S residues indicate the G242A and S261A substitutions (this study). The references indicated by superscripts are as described in the legend to panel A.

FIG. 3.

Comparison of the kinetic parameters of CpdB of Pseudomonas sp. strain HI-70 towards long-chain cyclic ketones. C₁₁, C₁₂, C₁₃, C₁₅, and C₁₅₊₁ are cycloundecanone, cyclododecanone, cyclotridecanone, cyclopentadecanone, and 2-methycyclopentadecanone, respectively. The bars represent the K_m values in μM. Solid line, k_cat; dashed line, catalytic efficiency (k_cat/K_m).

FIG. 4.

The relative activities of purified CpdB toward a range of large and small cyclic ketones and bicyclic ketones. The specific activity (3.9 U/mg) toward cyclopentadecanone (substrate 4) is taken as 100%. n.d., not determined.

FIG. 5.

Overlaid infrared spectra of a time course monitoring of cyclododecanone conversion to lauryl lactone catalyzed by CpdB using the ReactIR 4000. A quantity of 0.2 U of the purified enzyme was used. · · · · · , 0 min; - · - · , 5 min; — —, 10 min; - - -, 20 min; —, 60 min.