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

Abstract

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.

The Purification and Properties of 4‐Hydroxyisophthalate Hydroxylase from Pseudomonas putida NCIB 9866

“…A comparable value of 4.5 x lo-' M was found for the Kd of an FMNcontaining enzyme, the NADH dehydrogenase component of the camphor-lactonizing system, and this protein loses all bound FMN during purification [37]. The Kd for cyclohexanone oxygenase-FAD was calculated as 4 x lo-* M and was considered to be just compatible with retention of flavin during purification [38]. Thus it seems likely that the high Kd for 4-hydroxyisophthalate hydroxylase may be attributable to irreversible damage to the apoenzyme during its preparation which may also be reflected in the inability to regain full activity.…”

Application of a thermostable Baeyer–Villiger monooxygenase for the synthesis of branched polyester precursors

“…BVMOs display a broad substrate scope,4, 5 which include cyclic ketones with a heteroatom,6, 7 macrocyclic ketones,8, 9, 10, 11 and substituted derivatives of various ring size (for example, terpene‐based substrates12, 13). 14, 15 BVMOs are also especially interesting in the synthesis of branched lactones, from which branched aliphatic polyesters with low glass transition temperatures can be made 16, 17, 18, 19, 20, 21, 22, 23, 24, 25…”

“…low thermostability and poor solvent tolerance), substrate and product inhibition as well as the regeneration of their NAD(P)H co‐factor 5. Although AcCHMO is the most widely studied BVMO,14 it displays poor solvent tolerance as well as low thermostability which limits its use as a biocatalyst 35, 36. A robust cyclohexanone monooxygenase from Thermocrispum municipale DSM 44069 (TmCHMO; EC 1.14.13.22) has recently been discovered 35.…”

Metabolism of Monoterpenes

“…(1,9,19). If subsequent steps in the metabolism of the lactone are oxidative, it seems likely that the lactone would initially undergo activation to the corresponding hydroxy acyl CoA ester.…”