Purification and characterization of a Baeyer-Villiger mono-oxygenase from Rhodococcus erythropolis DCL14 involved in three different monocyclic monoterpene degradation pathways.
A Baeyer-Villiger mono-oxygenase (BVMO), catalysing the NADPH- and oxygen-dependent oxidation of the monocyclic monoterpene ketones 1-hydroxy-2-oxolimonene, dihydrocarvone and menthone, was purified to homogeneity from Rhodococcus erythropolis DCL14. Monocyclic monoterpene ketone mono-oxygenase (MMKMO) is a monomeric enzyme of molecular mass 60 kDa. It contains 1 mol of FAD/monomer as the prosthetic group. The N-terminal amino acid sequence showed homology with many other NADPH-dependent and FAD-containing (Type 1) BVMOs. Maximal enzyme activity was measured at pH 9 and 35 degrees C. MMKMO has a broad substrate specificity, catalysing the lactonization of a large number of monocyclic monoterpene ketones and substituted cyclohexanones. The natural substrates 1-hydroxy-2-oxolimonene, dihydrocarvone and menthone were converted stoichiometrically into 3-isopropenyl-6-oxoheptanoate (the spontaneous rearrangement product of the lactone formed by MMKMO), 4-isopropenyl-7-methyl-2-oxo-oxepanone and 7-isopropyl-4-methyl-2-oxo-oxepanone respectively. The MMKMO-catalysed conversion of iso-dihydrocarvone showed an opposite regioselectivity to that of dihydrocarvone; in this case, 6-isopropenyl-3-methyl-2-oxo-oxepanone was formed as the product. MMKMO converted all enantiomers of the natural substrates with almost equal efficiency. MMKMO is involved in the conversion of the monocyclic monoterpene ketone intermediates formed in the degradation pathways of all stereoisomers of three different monocyclic monoterpenes, i.e. limonene, (dihydro)carveol and menthol.
Van Der Werf
M J
MJ
Division of Industrial Microbiology, Department of Food Technology and Nutritional Sciences, Wageningen University, 6700 EV Wageningen, The Netherlands. vanderWerf@voeding.tno.nl
D000447:Aldehydes / Q000378:metabolism; D000595:Amino Acid Sequence; D001673:Biodegradation, Environmental; D002384:Catalysis / Q000187:drug effects; D003512:Cyclohexanones / Q000378:metabolism; D004790:Enzyme Induction; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D008610:Menthol; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010105:Oxygenases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D012240:Rhodococcus / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013057:Spectrum Analysis; D013237:Stereoisomerism; D013379:Substrate Specificity; D013696:Temperature; D013729:Terpenes / Q000378:metabolism*
D000447:Aldehydes; D003512:Cyclohexanones; D004791:Enzyme Inhibitors; D008670:Metals; D013729:Terpenes; D005182:Flavin-Adenine Dinucleotide; D008610:Menthol; C019466:menthone; D010105:Oxygenases; C409322:monocyclic monoterpeneketone mono-oxygenase