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. 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 (NH4)2SO4 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. 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.‐). 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. The N. globerula enzyme has a pH optimum of 8.4, apparent Km 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 Km 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. 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. These enzymes bear a strong resemblance to several of the monooxygenases that hydroxylate aromatic compounds.

Acinetobacter NCIB 9871 was isolated by elective culture on cyclohexanol and grows with this compound as sole source of carbon. It displays a restricted growth spectrum, being unable to grow on a wide range of alternative alicyclic alcohols and ketones. Cyclohexanol-grown cells oxidize the growth substrate at a rate of 230 pl of 0 2 / h per mg dry wt with the consumption of 5.65 pmol of 02/ pmol substrate. Cyclohexanone is oxidized at a similar rate with the consumption of 4.85 pmol of 02/pmol. l-Oxa-2-oxocycloheptane and 6-hydroxyhexanoate are both oxidized at the same slow rate of 44 pl of Oz/h per mg dry wt and adipate is not oxidized.Studies with cell extracts reveal the presence of inducible dehydrogenases for cyclohexanol, 6-hydroxyhexanoate and 6-oxohexanoate and a monooxygenase, that in conjunction with a lactonase converts cyclohexanone to 6-hydroxyhexanoate. The monooxygenase is therefore presumed to be of the lactone-forming type and the pathway for conversion of cyclohexanol to adipate ; cyclohexanol + cyclohexanone + 1 -oxa-2-oxocycloheptane -+ 6-hydroxyhexanoate -+ 6-oxohexanoate -+ adipate ; for which key intermediates have been identified chromatographically, is identical with the route for the oxidation of cyclohexanol by Nocardia globerula CL1.Though the metabolism of aromatic compounds has been the subject of intensive research the cycloalkanes have only recently received serious attention. Posternak et al. [l] reported that Acetobacter suboxydans growing with either isomer of cyclohexan-1 ,2-diol released 2-hydroxycyclohexan-l-one into the growth medium and Yugari [2] showed that a species of Pseudomonas oxidized trans-cyclohexan-1 ,2-diol to cyclohexan-1,2-dione which was then cleaved by the action of a hydrolase to form 6-oxbhexanoate. The metabolism of cyclohexanol by a strain of Nocardia has been shown to involve the pathway cyclohexanol + cyclohexanone + l-oxa-2-oxocycloheptane (a lactone) + 6-hydroxyhexanoate -+ adipate [3] and it has been suggested that the adipate is further metabolized by P-oxidation. Shaw [4] reported the oxidation of cyclopentanone to glutarate by several gram negative bacteria and the involvement of a lactone-forming mixed-function oxygenase in the metabolism of cyclopentanol by Pseudomonas NCIB 9872 has been described [5]. A report of the involvement of an hydroxylase in the metabolism of cyclohexanone by a strain of Nocardia has recently appeared [6] with the implied

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