54 -dependent regulatory elements in front of the divergently transcribed cpnB and cpnC genes supports the notion that cpnR is a regulatory gene of the NtrC type. Knowledge of the nucleotide sequence of the cpn genes was used to construct isopropyl-␤-thio-D-galactoside-inducible clones of Escherichia coli cells that overproduce the five enzymes of the cpn pathway. The substrate specificities of CpnA and CpnB were studied in particular to evaluate the potential of these enzymes and establish the latter recombinant strain as a bioreagent for Baeyer-Villiger oxidations. Although frequently nonenantioselective, cyclopentanone 1,2-monooxygenase was found to exhibit a broader substrate range than the related cyclohexanone 1,2-monooxygenase from Acinetobacter sp. strain NCIMB 9871. However, in a few cases opposite enantioselectivity was observed between the two biocatalysts.Pseudomonas sp. strain NCIMB 9872, which is capable of growth on 0.1% cyclopentanol as sole carbon source, was isolated from a freshwater stream in Illinois by P. J. Chapman some three decades ago. Trudgill and coworkers (27) used this strain as a prototype organism to establish the biochemical pathway of cyclopentanol metabolism shown in Fig. 1. For reason of utility as a Baeyer-Villiger (BV) biocatalyst and its mechanistic aspects (2,8,13,58,71), the second biochemical step catalyzed by cyclopentanone 1,2-monooxygenase (CPMO) has been most extensively studied in this organism. The conversion of cyclopentanone to 5-valerolactone is NADPH and flavin adenine dinucleotide (FAD) dependent, and CPMO has been purified to near homogeneity. It is a tetramer of subunit molecular weight 50,000 to 54,000 (8,66). Assay of the FAD/ protein ratio gave values of 2 to 4 molecules of FAD bound to each tetrameric enzyme molecule (28,66). An N-terminal 29-amino-acid sequence of CPMO has been determined (8).A chemical BV oxidation is the transformation of ketones into esters or of cyclic ketones into lactones by peracids, such as 3-chloroperbenzoic acid. This century-old reaction (for reviews, see references 54 and 63) continues to attract interest not only in broadening the spectrum of applications (ranging from the synthesis of steroids, antibiotics, and pheromones to the synthesis of monomers for polymerization, etc.) but also in developing oxidants that are more chemoselective and efficient and thus result in more product than waste (20).The biological BV reactions catalyzed by BV monooxygenases (BVMOs) offer the prospect of eco-efficient chemical transformations in whole cells or improved expression systems via cloning. Moreover, BVMOs can provide products in optically active form which are difficult to obtain by other strategies (for reviews, see references 55, 61, 71, and 74). Notable ste-* Corresponding author. Mailing address:

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 ϳ60 to 147 amino acids. The 64-kDa CPDMO enzyme was purified to apparent homogeneity, providing a specific activity of 3.94 mol/min/mg protein and a 20% yield. CPDMO is monomeric and NADPH dependent and contains ϳ1 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 M versus K m ‫؍‬ 24 M). 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 ␤-tetralone. CPDMO has the highest affinity (K m ‫؍‬ 5.8 M) and the highest catalytic efficiency (k cat /K m ratio of 7.2 ؋ 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.

Whole cells of an Escherichia coli strain that overexpresses Acinetobacter sp. NCIB 9871 cyclohexanone monooxygenase have been used for the Baeyer-Villiger oxidations of a variety of 4-mono- and 4,4-disubstituted cyclohexanones. In cases where comparisons were possible, this new biocatalytic reagent provided lactones with chemical yields and optical purities that were comparable to those obtained from the purified enzyme or a strain of bakers' yeast that expresses the same enzyme. The efficient production of cyclohexanone monooxygenase in the E. coli expression system (ca. 30% of total soluble protein) allowed these oxidations to reach completion in approximately half the time required for the engineered bakers' yeast strain. Surprisingly, 4,4-disubstituted cyclohexanones were also accepted by the enzyme, and the enantioselectivities of these oxidations could be rationalized by considering the conformational energies of bound substrates along with the enzyme's intrinsic enantioselectivity. The enzyme expressed in E. coli cells also oxidized several 4-substituted cyclohexanones bearing polar substituents, often with high enantioselectivities. In the case of 4-iodocyclohexanone, the lactone was obtained in > 98% ee and its absolute configuration was assigned by X-ray crystallography. The crystal belongs to the monoclinic P2(1) space group with a = 5.7400(10), b = 6.1650(10), c = 11.377(2) A, b = 99.98(2) degrees, and Z = 2. Taken together, these results demonstrate the utility of an engineered bacterial strain in delivering useful chiral building blocks in an experimentally simple manner.