Steroid Monooxygenase of Rhodococcus rhodochrous: Sequencing of the Grenomic DNA, and Hyperexpression, Purification, and Characterization of the Recombinant Enzyme

Morii, S.; Sawamoto, S.; Yamauchi, Yoritaka; Miyamoto, M.; Iwami, Masafumi; Itagaki, Eiji

doi:10.1093/oxfordjournals.jbchem.a022494

Cited by 53 publications

(37 citation statements)

References 20 publications

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“…BP2 and Rhodococcus sp. strains Phi1 and Phi2 steroid monooxygenase from Rhodococcus rhodochrous (38), and an alkane monooxygenase of P. fluorescens DSM 50106 (31). In addition to these characterized BVMOs, the putative cyclohexanone monooxygenase from Rhodococcus sp.…”

Section: Discussionmentioning

confidence: 99%

Degradation of Alkyl Methyl Ketones by Pseudomonas veronii MEK700

et al. 2007

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Pseudomonas veronii MEK700 was isolated from a biotrickling filter cleaning 2-butanone-loaded waste air. The strain is able to grow on 2-butanone and 2-hexanol. The genes for degradation of short chain alkyl methyl ketones were identified by transposon mutagenesis using a newly designed transposon, mini-Tn5495, and cloned in Escherichia coli. DNA sequence analysis of a 15-kb fragment revealed three genes involved in methyl ketone degradation. The deduced amino acid sequence of the first gene, mekA, had high similarity to BaeyerVilliger monooxygenases; the protein of the second gene, mekB, had similarity to homoserine acetyltransferases; the third gene, mekR, encoded a putative transcriptional activator of the AraC/XylS family. The three genes were located between two gene groups: one comprising a putative phosphoenolpyruvate synthase and glycogen synthase, and the other eight genes for the subunits of an ATPase. Inactivation of mekA and mekB by insertion of the mini-transposon abolished growth of P. veronii MEK700 on 2-butanone and 2-hexanol. The involvement of mekR in methyl ketone degradation was observed by heterologous expression of mekA and mekB in Pseudomonas putida. A fragment containing mekA and mekB on a plasmid was not sufficient to allow P. putida KT2440 to grow on 2-butanone. Not until all three genes were assembled in the recombinant P. putida was it able to use 2-butanone as carbon source. The Baeyer-Villiger monooxygenase activity of MekA was clearly demonstrated by incubating a mekB transposon insertion mutant of P. veronii with 2-butanone. Hereby, ethyl acetate was accumulated. To our knowledge, this is the first time that ethyl acetate by gas chromatographic analysis has been definitely demonstrated to be an intermediate of MEK degradation. The mekB-encoded protein was heterologously expressed in E. coli and purified by immobilized metal affinity chromatography. The protein exhibited high esterase activity towards short chain esters like ethyl acetate and 4-nitrophenyl acetate.

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Section: Discussionmentioning

confidence: 99%

Degradation of Alkyl Methyl Ketones by Pseudomonas veronii MEK700

et al. 2007

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“…Cloning, Expression, and Purification-The gene coding for STMO from R. rhodochrous (8) was cloned into a Champion TM pET-vector (Invitrogen) for overexpression in Escherichia coli. The resulting protein contained a N-terminal His 6 -tagged SUMO, which can be removed by SUMO protease.…”

Section: Methodsmentioning

confidence: 99%

“…In 1999, steroid monooxygenase (STMO) from Rhodococcus rhodochrous was the second BVMO for which the gene was cloned and used for heterologous expression (8). However, so far no subsequent studies on this bacterial steroid degrading monooxygenase have been reported.…”

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confidence: 99%

“…This enzyme is capable of converting progesterone into progesterone acetate (Scheme 1). The interest for this enzyme stems from its high sequence homology with the most well characterized BVMOs with known crystal structures (8,17); STMO has 53% sequence identity with PAMO from Thermobifida fusca, 45% with CHMO from Rhodococcus sp. strain HI-31, and 43% with OTEMO from Pseudomonas putida.…”

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Exploring the Structural Basis of Substrate Preferences in Baeyer-Villiger Monooxygenases

Franceschini

Beek

Pennetta

et al. 2012

Journal of Biological Chemistry

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Background: Bacterial steroid monooxygenase degrades progesterone. Results: The crystallographic and mutagenesis analysis outline a robust active-site scaffold, capable of performing BaeyerVilliger oxidations on chemically diverse molecules. Conclusion: This and related enzymes represent a fascinating case for the comparative evaluation of user tailored protein engineering with enzyme variants arising through evolution. Significance: These findings highlight the biocatalytic potential of Baeyer-Villiger monooxygenases.

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“…Sequence comparison using the BLASTX program against the nonredundant GenBank database showed that the two Brevibacterium cyclohexanone monooxygenases are part of the large family of flavin-dependent monooxygenases. Sequences with the highest similarity are the Acinetobacter cyclohexanone monooxygenase (A28550) and the Rhodococcus steroid monooxygenase (AB010439) (3,24). The activities of both proteins have been characterized biochemically.…”

Section: Figmentioning

confidence: 99%

Simultaneous Identification of Two Cyclohexanone Oxidation Genes from an Environmental Brevibacterium Isolate Using mRNA Differential Display

et al. 2000

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The technique of mRNA differential display was used to identify simultaneously two metabolic genes involved in the degradation of cyclohexanone in a new halotolerant Brevibacterium environmental isolate. In a strategy based only on the knowledge that cyclohexanone oxidation was inducible in this strain, the mRNA population of cells exposed to cyclohexanone was compared to that of control cells using reverse transcription-PCR reactions primed with a collection of 81 arbitrary oligonucleotides. Three DNA fragments encoding segments of flavin monooxygenases were isolated with this technique, leading to the identification of the genes of two distinct cyclohexanone monooxygenases, the enzymes responsible for the oxidation of cyclohexanone. Each monooxygenase was expressed in Escherichia coli and characterized. This work validates the application of mRNA differential display for the discovery of new microbial metabolic genes.It is now widely accepted that the diversity of microorganisms extends far beyond the few thousand species in culture collections (15). This diversity of microbes and their metabolism constitutes a vast source of enzymes and genes for biotechnology applications. The identification of useful metabolic genes has traditionally proceeded either through a direct genetic approach or by the reverse genetics approach, starting with the purification of the enzyme of interest followed by identification of its gene through the use of antibodies or amino acid sequence information obtained from the pure protein.Although both strategies are routinely used, they are often limited by technical problems. The direct genetic approach can be used only for organisms that have a developed genetic system or whose genes can be expressed in heterologous hosts. The reverse genetics approach requires purification of the protein of interest, which often takes a long time, and the successful amplification of a DNA probe from degenerate primers, a technique that sometimes fails. Recently mRNA techniques have made it possible to access regulated genes directly without the purification of their gene products and in the absence of a genetic system. These approaches are based on comparison of the mRNA population between two cultures or tissues and identification of the subset of genes whose mRNA is more abundant under conditions of induction. These techniques rely on the hybridization of labeled mRNAs onto arrays of DNA on membranes (4) or DNA microarrays (9), large-scale sample sequencing of expressed sequence tag libraries (28), or the sampling of mRNA by the production of randomly amplified DNA fragments by reverse transcription (RT) followed by PCR (RT-PCR) (19,20,35). Because it can easily be done by individual scientists at low cost, the latter approach has been used extensively since it was first described.Two variations of this RT-PCR method have been published. The first, called differential display (DD) (19,20), begins with the synthesis of cDNAs by RT of mRNA using a poly(dT) primer that hybridizes to the poly(A) tail o...

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Steroid Monooxygenase of Rhodococcus rhodochrous: Sequencing of the Grenomic DNA, and Hyperexpression, Purification, and Characterization of the Recombinant Enzyme

Cited by 53 publications

References 20 publications

Degradation of Alkyl Methyl Ketones by Pseudomonas veronii MEK700

Degradation of Alkyl Methyl Ketones by Pseudomonas veronii MEK700

Exploring the Structural Basis of Substrate Preferences in Baeyer-Villiger Monooxygenases

Simultaneous Identification of Two Cyclohexanone Oxidation Genes from an Environmental Brevibacterium Isolate Using mRNA Differential Display

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