Department of Bio- and Environmental Sciences, International Graduate School of Zittau, Markt 23, 02763, Zittau, Germany.
AMB Express. 2013 Jan 17;3(1):5. doi: 10.1186/2191-0855-3-5.
Aromatic peroxygenase (APO) is an extracellular enzyme produced by the agaric basidiomycete Agrocybe aegerita that catalyzes diverse peroxide-dependent oxyfunctionalization reactions. Here we describe the oxygenation of the unactivated aromatic ring of benzene with hydrogen peroxide as co-substrate. The optimum pH of the reaction was around 7 and it proceeded via an initial epoxide intermediate that re-aromatized in aqueous solution to form phenol. Identity of the epoxide intermediate as benzene oxide was proved by a freshly prepared authentic standard using GC-MS and LC-MS analyses. Second and third [per]oxygenation was also observed and resulted in the formation of further hydroxylation and following [per]oxidation products: hydroquinone and p-benzoquinone, catechol and o-benzoquinone as well as 1,2,4-trihydroxybenzene and hydroxy-p-benzoquinone, respectively. Using H218O2 as co-substrate and ascorbic acid as radical scavenger, inhibiting the formation of peroxidation products (e.g., p-benzoquinone), the origin of the oxygen atom incorporated into benzene or phenol was proved to be the peroxide. Apparent enzyme kinetic constants (kcat, Km) for the peroxygenation of benzene were estimated to be around 8 s-1 and 3.6 mM. These results raise the possibility that peroxygenases may be useful for enzymatic syntheses of hydroxylated benzene derivatives under mild conditions.
芳香过氧化物酶（APO）是一种由金针菇担子菌产生的细胞外酶，可催化多种依赖过氧化物的氧化官能化反应。在这里，我们描述了用过氧化氢作为共底物对苯的未激活芳环进行的氧化。反应的最适 pH 值约为 7，它通过初始环氧化物中间体进行，该中间体在水溶液中重新芳构化为苯酚。通过使用 GC-MS 和 LC-MS 分析新制备的苯氧化物标准品，证明了环氧化物中间体为苯氧化物。还观察到第二和第三 [过] 氧合作用，导致进一步的羟化和随后的 [过] 氧化产物的形成：氢醌和对苯醌、儿茶酚和邻苯醌以及 1,2,4-三羟基苯和羟基-对苯醌，分别。使用 H218O2 作为共底物和抗坏血酸作为自由基清除剂，抑制过氧化物产物（例如对苯醌）的形成，证明掺入苯或苯酚中的氧原子来自过氧化物。苯过氧合的表观酶动力学常数（kcat，Km）估计约为 8 s-1 和 3.6 mM。这些结果表明过氧化物酶在温和条件下可能对羟化苯衍生物的酶合成有用。
