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Purification and characterization of a Baeyer-Villiger mono-oxygenase from Rhodococcus erythropolis DCL14 involved in three different monocyclic monoterpene degradation pathways

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Author: Werf, M.J. van der
Type:article
Date:2000
Institution: Centraal Instituut voor Voedingsonderzoek TNO
Source:Biochemical Journal, 3, 347, 693-701
Identifier: 72166
doi: doi:10.1042/0264-6021:3470693
Keywords: Nutrition · Carvone · Flavoprotein · Limonene · Menthol · Regioselectivity · Bacterial enzyme · Carvone · Cyclohexanone derivative · Heptanoic acid derivative · Limonene · Menthone · Terpene · Unspecific monooxygenase · Amino acid sequence · Amino terminal sequence · Biodegradation · Enzyme activity · Enzyme analysis · Enzyme purification · Enzyme specificity · Enzyme structure · Molecular weight · Nonhuman · Priority journal · Rhodococcus · Sequence homology · Stereochemistry · Aldehydes · Amino Acid Sequence · Biodegradation, Environmental · Catalysis · Cyclohexanones · Enzyme Induction · Enzyme Inhibitors · Flavin-Adenine Dinucleotide · Hydrogen-Ion Concentration · Menthol · Metals · Molecular Sequence Data · Molecular Weight · Oxygenases · Rhodococcus · Sequence Alignment · Sequence Homology, Amino Acid · Spectrum Analysis · Stereoisomerism · Substrate Specificity · Temperature · Terpenes

Abstract

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°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-oxo-limonene, 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. Chemicals/CAS: Aldehydes; Cyclohexanones; Enzyme Inhibitors; Flavin-Adenine Dinucleotide, 146-14-5; Menthol, 1490-04-6; menthone, 89-80-5; Metals; monocyclic monoterpeneketone mono-oxygenase, EC 1.14.13.-; Oxygenases, EC 1.13.-; Terpenes