Zhang Xiaodong, Hu Yun, Peng Wei, Gao Chenghua, Xing Qiong, Wang Binju, Li Aitao
State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan, China.
State Key Laboratory of Physical Chemistry of Solid Surfaces, Collaborative Innovation Center of Chemistry for Energy Materials, National Engineering Laboratory for Green Chemical Productions of Alcohols, Ethers and Esters, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, China.
Front Chem. 2021 Feb 18;9:649000. doi: 10.3389/fchem.2021.649000. eCollection 2021.
Cytochrome P450 enzyme CYP109B1 is a versatile biocatalyst exhibiting hydroxylation activities toward various substrates. However, the regio- and stereoselective steroid hydroxylation by CYP109B1 is far less explored. In this study, the oxidizing activity of CYP109B1 is reconstituted by coupling redox pairs from different sources, or by fusing it to the reductase domain of two self-sufficient P450 enzymes P450RhF and P450BM3 to generate the fused enzyme. The recombinant expressing necessary proteins are individually constructed and compared in steroid hydroxylation. The ferredoxin reductase (Fdr_0978) and ferredoxin (Fdx_1499) from is found to be the best redox pair for CYP109B1, which gives above 99% conversion with 73% 15β selectivity for testosterone. By contrast, the rest ones and the fused enzymes show much less or negligible activity. With the aid of redox pair of Fdr_0978/Fdx_1499, CYP109B1 is used for hydroxylating different steroids. The results show that CYP109B1 displayed good to excellent activity and selectivity toward four testosterone derivatives, giving all 15β-hydroxylated steroids as main products except for 9 (10)-dehydronandrolone, for which the selectivity is shifted to 16β. While for substrates bearing bulky substitutions at C17 position, the activity is essentially lost. Finally, the origin of activity and selectivity for CYP109B1 catalyzed steroid hydroxylation is revealed by computational analysis, thus providing theoretical basis for directed evolution to further improve its catalytic properties.
细胞色素P450酶CYP109B1是一种多功能生物催化剂，对多种底物具有羟基化活性。然而，CYP109B1对甾体的区域和立体选择性羟基化研究较少。在本研究中，通过偶联不同来源的氧化还原对，或将其与两种自给自足的P450酶P450RhF和P450BM3的还原酶结构域融合以生成融合酶，来重建CYP109B1的氧化活性。分别构建表达所需蛋白质的重组体，并比较它们在甾体羟基化中的作用。发现来自[具体来源未给出]的铁氧还蛋白还原酶（Fdr_0978）和铁氧还蛋白（Fdx_1499）是CYP109B1的最佳氧化还原对，其对睾酮的转化率高于99%，15β选择性为73%。相比之下，其他氧化还原对和融合酶的活性则低得多或可忽略不计。借助Fdr_0978/Fdx_1499氧化还原对，CYP109B1用于羟基化不同的甾体。结果表明，CYP109B1对四种睾酮衍生物表现出良好到优异的活性和选择性，除9(10)-脱氢诺龙外，所有产物均以15β-羟基化甾体为主，其选择性转移至16β。而对于在C17位带有大体积取代基的底物，活性基本丧失。最后，通过计算分析揭示了CYP109B1催化甾体羟基化的活性和选择性来源，从而为定向进化以进一步改善其催化性能提供了理论依据。
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