The Purification and Properties of Cyclohexanone Oxygenase from Nocardia globerula CL1 and Acinetobacter NCIB 9871

1 Cyclohexanone oxygenases from Nocardia globerula CL1 and Acinetobacter NCIB 9571 have been purified 12‐fold and 35‐fold respectively and each gives a single symmetrical sedimentation peak in the ultracentrifuge and a single protein band on 2.25 nm average pore radius polyacrylamide gels. 2 The enzyme from N. globerula has a molecular weight of 53000 while that from Acinetobacter has a molecular weight of about 59000. Each is a single polypeptide chain with one mole of bound FAD per mole of protein that does not dissociate during purification. Acidification of the Acinetobacter enzyme in the presence of (NH 4 ) 2 SO 4 releases the bound FAD and yields native apoenzyme from which the active holoenzyme can be reconstituted. The apparent dissociation constant for the FAD is 40 nM. 3 The near unitary stoichiometry of cyclohexanone, NADPH and oxygen consumption is typical of mixed function oxygenases with external electron donors. The oxygenated product has been identified as 1‐oxa‐2‐oxocycloheptane thus placing these enzymes in the small group of lactone and ester‐forming oxygenases. Their correct systematic name is cyclohexanone. NADPH: oxygen oxidoreductase (1,2‐lactonizing) (EC 1.14.13.‐). 4 A functionally essential sulfhydryl group is present at the catalytic centre of both enzymes but there is no reliable indication from inhibitor studies that they contain any functional metal ion. The three titratable sulfhydryl groups of the Acinetobacter enzyme are not equivalent since reaction with one of them selectively inhibits catalytic activity. Protection against sulfhydryl active agents is afforded by NADPH but not by cyclohexanone. 5 The N. globerula enzyme has a pH optimum of 8.4, apparent K m values of 1.56 μM and 31.3 μM for cyclohexanone and NADPH respectively and a catalytic centre activity of 1018 ml substrate transformed × mol enzyme −1 × min −1 . The Acinetobacter enzyme has a pH optimum of 9.0, apparent K m values of 6.9 tM and 17.8 μM and a catalytic centre activity of 1390 mol × mol enzyme −1 × min −1. Both enzymes display absolute specificity for electron donor which contrasts with the broad specificity for ketone substrate. 6 An enzyme‐cyclohexanone complex has been detected by difference spectroscopy only in the case of the Nocardia enzyme. Rapid reduction of the enzyme‐bound FAD occurs upon addition of NADPH in the absence of cyclohexanone. Titration of enzyme with NADPH under anaerobic conditions and anaerobic photoreduction in the presence of EDTA have not revealed the formation of any stable flavin semiquinones. 7 These enzymes bear a strong resemblance to several of the monooxygenases that hydroxylate aromatic compounds.