Characterization of an FMN‐containing cyclohexanone monooxygenase from a cyclohexane‐grown Xanthobacter sp.

Abstract

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 <0.5 μM and 12.5 μM 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). Copyright © 1989, Wiley Blackwell. All rights reserved