Characterization of an FMN-containing cyclohexanone monooxygenase from a cyclohexane-grown Xanthobacter sp.
A soluble cyclohexanone monooxygenase was purified 16.1-fold to homogeneity from a Xanthobacter sp. grown upon cyclohexane as sole source of carbon and energy. The native enzyme is a 50-kDa single polypeptide chain associated with FMN rather than FAD as flavin prosthetic group in a 1:1 stoichiometric relationship. The monooxygenase catalyses the transformation of cyclohexanone to the lactone 1-oxa-2-oxocycloheptane in an oxygen ring insertion reaction. Only related cycloalkanone substrates are accepted for oxygenation, no activity is shown towards straight-chain alkanones. Enzyme activity is strongly inhibited by sulphydryl-reactive agents, but is relatively insensitive to metal chelators, electron transport inhibitors and the metal ions Fe3+ and Cu2+. Cyclohexanone monooxygenase has Km values for cyclohexanone and NADPH of less than 0.5 microM and 12.5 microM respectively. Kinetic investigations under steady-state conditions demonstrate that the flavoprotein prosthetic group, FMN, is involved in the monooxygenase catalytic mechanism. The systematic name for the enzyme is cyclohexanone, NADPH:oxygen oxidoreductase (6-hydroxylating, 1,2-lactonizing) (EC 1.14.13.22).
Trower
M K
MK
Department of Life Sciences, Trent Polytechnic, Nottingham, England.
Buckland
R M
RM
Griffin
M
M
D002845:Chromatography; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D005486:Flavin Mononucleotide / Q000032:analysis*; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology*; D006882:Hydroxyapatites; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000302:isolation & purification / Q000378:metabolism*; D013053:Spectrophotometry; D013379:Substrate Specificity
D006882:Hydroxyapatites; D046911:Macromolecular Substances; D005486:Flavin Mononucleotide; D017886:Durapatite; D010105:Oxygenases; C019961:cyclohexanone oxygenase