Department of Life Science and Biochemical Engineering, Sunmoon University, Asan 31460, Republic of Korea.
Chemical Biology Research Center, Korea Research Institute of Bioscience and Biotechnology, Ochang-eup, Chungbuk 28116, Republic of Korea.
J Microbiol Biotechnol. 2021 Mar 28;31(3):464-474. doi: 10.4014/jmb.2010.10020.
Bacterial cytochrome P450 (CYP) enzymes are responsible for the hydroxylation of diverse endogenous substances with a heme molecule used as a cofactor. This study characterized two CYP154C3 proteins from sp. W2061 (CYP154C3-1) and sp. KCCM40643 (CYP154C3-2). The enzymatic activity assays of both CYPs conducted using heterologous redox partners' putidaredoxin and putidaredoxin reductase showed substrate flexibility with different steroids and exhibited interesting product formation patterns. The enzymatic characterization revealed good activity over a pH range of 7.0 to 7.8 and the optimal temperature range for activity was 30 to 37°C. The major product was the C16-hydroxylated product and the kinetic profiles and patterns of the generated hydroxylated products differed between the two enzymes. Both enzymes showed a higher affinity toward progesterone, with CYP154C3-1 demonstrating slightly higher activity than CYP154C3-2 for most of the substrates. Oxidizing agents (diacetoxyiodo) benzene (PIDA) and hydrogen peroxide (HO) were also utilized to actively support the redox reactions, with optimum conversion achieved at concentrations of 3 mM and 65 mM, respectively. The oxidizing agents affected the product distribution, influencing the type and selectivity of the CYP-catalyzed reaction. Additionally, CYP154C3s also catalyzed the C-C bond cleavage of steroids. Therefore, CYP154C3s may be a good candidate for the production of modified steroids for various biological uses.
细菌细胞色素 P450（CYP）酶负责将各种内源性物质羟化，其辅因子为血红素分子。本研究对来自 sp. W2061（CYP154C3-1）和 sp. KCCM40643（CYP154C3-2）的两种 CYP154C3 蛋白进行了表征。使用异源氧化还原伴侣——putidaredoxin 和 putidaredoxin reductase 进行的这两种 CYP 的酶活性测定显示出对不同类固醇的底物灵活性，并表现出有趣的产物形成模式。酶特性研究表明，在 pH 值为 7.0 至 7.8 的范围内具有良好的活性，最适活性温度范围为 30 至 37°C。主要产物是 C16-羟化产物，两种酶生成的羟化产物的动力学特征和模式不同。两种酶对孕酮都表现出较高的亲和力，对于大多数底物，CYP154C3-1 的活性略高于 CYP154C3-2。还利用氧化剂（二乙酰氧基碘苯）（PIDA）和过氧化氢（HO）来积极支持氧化还原反应，最佳转化率分别在 3 mM 和 65 mM 时达到。氧化剂会影响产物分布，影响 CYP 催化反应的类型和选择性。此外，CYP154C3 还催化类固醇的 C-C 键断裂。因此，CYP154C3 可能是用于各种生物用途的修饰类固醇生产的良好候选者。
