Functional expression, purification, and characterization of the recombinant Baeyer-Villiger monooxygenase MekA from Pseudomonas veronii MEK700.
For the investigation of the NADPH-dependent Baeyer-Villiger monooxygenase MekA from Pseudomonas veronii MEK700, the encoding gene mekA with a C-terminal strep-tag was cloned and expressed under the control of a L: -rhamnose inducible promoter from Escherichia coli. The mekA gene was found by analyzing the methylethylketone (MEK) degradation pathway by Onaca et al. J Bacteriol 189:3759-3767, 2007. Sequence analysis of the corresponding protein, which catalyzes the Baeyer-Villiger oxidation of MEK to ethyl acetate, showed two binding sites (Rossman-fold motifs) for cofactors NAD(P)H and FAD. Although expression of mekA resulted in large amounts of inclusion bodies compared to soluble protein, high amounts of purified and active MekA were obtained by affinity chromatography. The substrate spectrum of MekA was investigated with purified enzyme and whole cells using a variety of aliphatic, aromatic, and cyclic ketones including four chiral substrates. The specific activity of MekA with MEK as substrate was determined to be 1.1 U/mg protein. K (M) values were determined for MEK and the cofactors NADPH and NADH to be 6, 11, and 29 microM, respectively.
Völker
Anne
A
Institute of Industrial Genetics, Stuttgart University, Stuttgart, Germany.
Kirschner
Anett
A
Bornscheuer
Uwe T
UT
Altenbuchner
Josef
J
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002849:Chromatography, Gas; D003001:Cloning, Molecular; D003067:Coenzymes / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010105:Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D003067:Coenzymes; D011994:Recombinant Proteins; D010105:Oxygenases