National Engineering Laboratory for Industrial Enzymes and Tianjin Engineering Center for Biocatalytic Technology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China.
University of Chinese Academy of Sciences, Beijing, China.
Appl Environ Microbiol. 2019 Aug 29;85(18). doi: 10.1128/AEM.01182-19. Print 2019 Sep 15.
In this study, we identified two P450 enzymes (CYP5150AP3 and CYP5150AN1) from NBRC 6298 by combination of transcriptome sequencing and heterologous expression in The biotransformation of 11-deoxycortisol and testosterone by whole cells coexpressing the and genes demonstrated that the CYP5150AP3 enzyme possessed steroidal 7β-hydroxylase activities toward these substrates, and the regioselectivity was dependent on the structures of steroidal compounds. CYP5150AN1 catalyzed the 2β-hydroxylation of 11-deoxycortisol. It is interesting that they display different regioselectivity of hydroxylation from that of their isoenzyme, CYP5150AP2, which possesses 19- and 11β-hydroxylase activities. The steroidal hydroxylases CYP5150AP3 and CYP5150AN1 together with the previously characterized CYP5150AP2 belong to the CYP5150A family of P450 enzymes with high amino acid sequence identity, but they showed completely different regioselectivities toward 11-deoxycortisol, suggesting the regioselectivity diversity of steroidal hydroxylases of CYP5150 family. They are also distinct from the known bacterial and fungal steroidal hydroxylases in substrate specificity and regioselectivity. Biocatalytic hydroxylation is one of the important transformations for the functionalization of steroid nucleus rings but remains a very challenging task in organic synthesis. These hydroxylases are useful additions to the toolbox of hydroxylase enzymes for the functionalization of steroids at various positions.
在这项研究中，我们通过转录组测序和异源表达相结合，从 NBRC 6298 中鉴定出两种 P450 酶（CYP5150AP3 和 CYP5150AN1）。 利用共表达 和 基因的全细胞对 11-去氧皮质醇和睾酮的生物转化表明，CYP5150AP3 酶对这些底物具有甾体 7β-羟化酶活性，且区域选择性取决于甾体化合物的结构。CYP5150AN1 催化 11-去氧皮质醇的 2β-羟化。有趣的是，它们显示出与同工酶 CYP5150AP2 不同的羟化区域选择性，CYP5150AP2 具有 19-和 11β-羟化酶活性。甾体羟化酶 CYP5150AP3 和 CYP5150AN1 与之前表征的 CYP5150AP2 一起属于 CYP5150A 家族的 P450 酶，它们具有高度的氨基酸序列同一性，但它们对 11-去氧皮质醇表现出完全不同的区域选择性，表明 CYP5150 家族甾体羟化酶的区域选择性多样性。它们与已知的细菌和真菌甾体羟化酶在底物特异性和区域选择性方面也有明显不同。生物催化羟化是甾体核环功能化的重要转化之一，但在有机合成中仍然是一项极具挑战性的任务。这些羟化酶是甾体在各种位置功能化的羟化酶工具包的有用补充。
