Ryerson C C, Ballou D P, Walsh C
Biochemistry. 1982 May 25;21(11):2644-55. doi: 10.1021/bi00540a011.
The bacterial flavoprotein monooxygenase carries out an oxygen insertion reaction on cyclohexanone, with ring expansion to form the seven-membered cyclic product epsilon-caprolactone, a transformation quite distinct from the phenol leads to catechol transformation carried out by the bacterial flavoprotein aromatic hydroxylases. Cyclohexanone oxygenase catalysis involves the four-electron of O2 at the expense of a two-electron oxidation of NADPH, concomitant with a two-electron oxidation of cyclohexanone to epsilon-caprolactone. NADPH oxidase activity is fully coupled with oxygen transfer to substrate. Steady-state kinetic assays demonstrate a ter-ter mechanism for this enzyme. Pre-steady-state kinetic assays demonstrate the participation of a 4a-hydroperoxyflavin intermediate during catalysis. In addition to its ketolactonizing activity, cyclohexanone oxygenase carries out S-oxygenation of thiane to thiane 1-oxide, a reaction which represents a nucleophilic displacement by the sulfur upon the terminal oxygen of the hydroperoxide. This is in contrast to cyclohexanone oxygenations where the flavin hydroperoxide acts as a nucleophile. In addition, a stable apoenzyme form is accessible and can be reconstituted with various FAD analogues with up to 100% recovery of enzyme activity. The accumulated results presented here support a Baeyer-Villiger rearrangement mechanism for the enzymatic oxygenation of cyclohexanone.
细菌黄素蛋白单加氧酶对环己酮进行氧插入反应，伴随着环扩张形成七元环状产物ε-己内酯，这种转化与细菌黄素蛋白芳香族羟化酶催化的苯酚生成邻苯二酚的转化截然不同。环己酮单加氧酶催化过程涉及O₂的四电子还原，以NADPH的两电子氧化为代价，同时伴随着环己酮两电子氧化生成ε-己内酯。NADPH氧化酶活性与向底物的氧转移完全偶联。稳态动力学分析表明该酶的反应机制为双底物双产物机制。预稳态动力学分析表明在催化过程中有4a-氢过氧化黄素中间体参与。除了其酮内酯化活性外，环己酮单加氧酶还能将噻烷S-氧化为噻烷1-氧化物，该反应代表硫对氢过氧化物末端氧的亲核取代。这与环己酮氧化反应不同，在环己酮氧化反应中黄素氢过氧化物充当亲核试剂。此外，可获得稳定的脱辅基酶形式，并能用各种FAD类似物进行重组，酶活性恢复率高达100%。此处呈现的累积结果支持环己酮酶促氧化的拜耳-维利格重排机制。
