ESI- and MALDI-MS Analysis of Cyclohexanone Monooxygenase from Acinetobacter NCIB 9871

Kneller, M.Byron; Cheesman, Matthew J.; Rettie, Allan E.

doi:10.1006/bbrc.2001.4664

Cited by 12 publications

(8 citation statements)

References 17 publications

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“…in biotransformation, in addition to producing lactones with very high enantioselectivities in asymmetric BV oxidations of a large number of ketones (38,48,62). Another notable aspect of overexpression of a clone of CHMO in E. coli was the sequence determination by mass spectrometry (40) that confirmed the DNA-predicted sequence reported by Iwaki et al (32). A correct CHMO sequence is important in view of its structure-function analysis, an exercise which has apparently begun in various laboratories (15,40,59).…”

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confidence: 79%

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Cloning and Characterization of a Gene Cluster Involved in Cyclopentanol Metabolism in Comamonas sp. Strain NCIMB 9872 and Biotransformations Effected by Escherichia coli -Expressed Cyclopentanone 1,2-Monooxygenase

Iwaki

Hasegawa

Wang

et al. 2002

Appl Environ Microbiol

124

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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:

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confidence: 79%

“…strain NCIMB 9871 (18,32,61). This facilitated the construction of clones of CHMO in heterologous systems such as Saccharomyces cerevisiae and Escherichia coli (6,15,22,38,40,48,59). The new whole-cell engineered systems circumvented the potentially pathogenic Acinetobacter sp.…”

mentioning

confidence: 99%

Cloning and Characterization of a Gene Cluster Involved in Cyclopentanol Metabolism in Comamonas sp. Strain NCIMB 9872 and Biotransformations Effected by Escherichia coli -Expressed Cyclopentanone 1,2-Monooxygenase

Iwaki

Hasegawa

Wang

et al. 2002

Appl Environ Microbiol

124

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“…[42] Recent mass spectrometry experiments proved that the sequence deposited by Iwaki et al is the correct CHMO sequence. [43] The CHMO gene encodes a 60.9 kDa protein appearing as monomer upon purification (Table 1). Recently, six other cyclohexanone monooxygenase genes were identified.…”

Section: Cyclohexanone Monooxygenase (Chmo)mentioning

confidence: 99%

Baeyer–Villiger Monooxygenases, an Emerging Family of Flavin‐Dependent Biocatalysts

Janssen²,

et al. 2003

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Abstract:Baeyer±Villiger monooxygenases (BVMOs) are flavoenzymes that catalyze a remarkably wide variety of oxidative reactions such as regioand enantioselective Baeyer±Villiger oxidations and sulfoxidations. Several of these conversions are difficult to achieve using chemical approaches. Due to their selectivity and catalytic efficiency, BVMOs are highly valuable biocatalysts for the synthesis of a broad range of fine chemicals. For a long time, only one member of this class of flavin-containing biocatalysts had been cloned and overexpressed which has limited their application for synthetic processes. Recently a number of new genes that encode BVMOs have been sequenced and overexpressed. In this paper the biocatalytic properties of recently cloned BVMOs are reviewed. Furthermore, the potential for obtaining novel BVMOs from sequenced genomes will be discussed.

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“…Examples of the latter are the classical CHMO of Acinetobacter sp. (8,22,29), CPMO of Comamonas sp. strain NCIMB 9872 (23), steroid monooxygenase (42), hydroxyacetophenone monooxygenase (HAPMO) (25), and phenylacetone monooxygenase (PAMO) (17,37).…”

Section: Resultsmentioning

confidence: 99%

“…strain NCIMB 9871 (22,29). HAPMO (640 amino acids), an aromatic monooxygenase of P. fluorescens ACB, is by far the largest BVMO, with an Nterminal extension of about 135 residues (25).…”

Section: Discussionmentioning

confidence: 99%

Pseudomonad Cyclopentadecanone Monooxygenase Displaying an Uncommon Spectrum of Baeyer-Villiger Oxidations of Cyclic Ketones

Iwaki

Wang

Große

et al. 2006

Appl Environ Microbiol

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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.

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ESI- and MALDI-MS Analysis of Cyclohexanone Monooxygenase from Acinetobacter NCIB 9871

Cited by 12 publications

References 17 publications

Cloning and Characterization of a Gene Cluster Involved in Cyclopentanol Metabolism in Comamonas sp. Strain NCIMB 9872 and Biotransformations Effected by Escherichia coli -Expressed Cyclopentanone 1,2-Monooxygenase

Cloning and Characterization of a Gene Cluster Involved in Cyclopentanol Metabolism in Comamonas sp. Strain NCIMB 9872 and Biotransformations Effected by Escherichia coli -Expressed Cyclopentanone 1,2-Monooxygenase

Baeyer–Villiger Monooxygenases, an Emerging Family of Flavin‐Dependent Biocatalysts

Pseudomonad Cyclopentadecanone Monooxygenase Displaying an Uncommon Spectrum of Baeyer-Villiger Oxidations of Cyclic Ketones

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