Department of Life Science and Biotechnology, Kansai University, Suita, Osaka, Japan.
Biosci Biotechnol Biochem. 2022 Jan 24;86(2):199-208. doi: 10.1093/bbb/zbab199.
Cytochrome P450 monooxygenases play important roles in metabolism. Here, we report the identification and biochemical characterization of P450CHC, a novel self-sufficient cytochrome P450, from cyclohexanecarboxylate-degrading Paraburkholderia terrae KU-64. P450CHC was found to comprise a [2Fe-2S] ferredoxin domain, NAD(P)H-dependent FAD-containing reductase domain, FCD domain, and cytochrome P450 domain (in that order from the N terminus). Reverse transcription-polymerase chain reaction results indicated that the P450CHC-encoding chcA gene was inducible by cyclohexanecarboxylate. chcA overexpression in Escherichia coli and recombinant protein purification enabled functional characterization of P450CHC as a catalytically self-sufficient cytochrome P450 that hydroxylates cyclohexanecarboxylate. Kinetic analysis indicated that P450CHC largely preferred NADH (Km = 0.011 m m) over NADPH (Km = 0.21 m m). The Kd, Km, and kcat values for cyclohexanecarboxylate were 0.083 m m, 0.084 m m, and 15.9 s-1, respectively. The genetic and biochemical analyses indicated that the physiological role of P450CHC is initial hydroxylation in the cyclohexanecarboxylate degradation pathway.
细胞色素 P450 单加氧酶在代谢中发挥重要作用。在这里，我们报告了一种新型自足细胞色素 P450 P450CHC 的鉴定和生化特性，它来自环己烷羧酸降解的 Paraburkholderia terrae KU-64。发现 P450CHC 由[2Fe-2S]铁氧还蛋白结构域、NAD(P)H 依赖性 FAD 结合还原酶结构域、FCD 结构域和细胞色素 P450 结构域（从 N 端开始）组成。逆转录聚合酶链反应结果表明，P450CHC 编码基因 chcA 可被环己烷羧酸诱导。在大肠杆菌中过表达 chcA 和重组蛋白纯化使 P450CHC 的功能特征得以实现，它是一种催化自足的细胞色素 P450，可羟基化环己烷羧酸。动力学分析表明，P450CHC 主要偏爱 NADH（Km = 0.011 m m）而不是 NADPH（Km = 0.21 m m）。环己烷羧酸的 Kd、Km 和 kcat 值分别为 0.083 m m、0.084 m m 和 15.9 s-1。遗传和生化分析表明，P450CHC 的生理作用是在环己烷羧酸降解途径中进行初始羟基化。
