Ocean College, Zhejiang University, Zhoushan, Zhejiang, China.
Kitasato Institute for Life Sciences, Kitasato University, Sagamihara, Kanagawa, Japan.
Appl Environ Microbiol. 2019 Nov 14;85(23). doi: 10.1128/AEM.01530-19. Print 2019 Dec 1.
Our previous study showed that CYP105D7, a substrate-promiscuous P450, catalyzes the hydroxylation of 1-deoxypentalenic acid, diclofenac, naringenin, and compactin. In this study, 14 steroid compounds were screened using recombinant cells harboring genes encoding CYP105D7 and redox partners (Pdx/Pdr, RhFRED, and FdxH/FprD), and the screening identified steroid A-ring 2β- and D-ring 16β-hydroxylation activity. Wild-type CYP105D7 was able to catalyze the hydroxylation of five steroids (testosterone, progesterone, 4-androstene-3,17-dione, adrenosterone, and cortisone) with low (<10%) conversion rates. Structure-guided site-directed mutagenesis of arginine residues around the substrate entrance and active site showed that the R70A and R190A single mutants and an R70A/R190A double mutant exhibited greatly enhanced conversion rates for steroid hydroxylation. For the conversion of testosterone in particular, the R70A/R190A mutant's / values increased 1.35-fold and the conversion rates increased significantly by almost 9-fold with high regio- and stereoselectivity. Molecular docking analysis revealed that when Arg70 and Arg190 were replaced with alanine, the volume of the substrate access and binding pocket increased 1.08-fold, which might facilitate improvement of the hydroxylation efficiency of steroids. Cytochrome P450 monooxygenases (P450s) are able to introduce oxygen atoms into nonreactive hydrocarbon compounds under mild conditions, thereby offering significant advantages compared to chemical catalysts. Promiscuous P450s with broad substrate specificity and reaction diversity have significant potential for applications in various fields, including synthetic biology. The study of the function, molecular mechanisms, and rational engineering of substrate-promiscuous P450s from microbial sources is important to fulfill this potential. Here, we present a microbial substrate-promiscuous P450, CYP105D7, which can catalyze hydroxylation of steroids. The loss of the bulky side chains of Arg70 and Arg190 in the active site and substrate entrance resulted in an up to 9-fold increase in the substrate conversion rate. These findings will support future rational and semirational engineering of P450s for applications as biocatalysts.
我们之前的研究表明，CYP105D7 是一种底物混杂的 P450，能够催化 1-去氧戊烯酸、双氯芬酸、柚皮素和考来替泊的羟化反应。在本研究中，使用含有 CYP105D7 和氧化还原伴侣（Pdx/Pdr、RhFRED 和 FdxH/FprD）基因的重组细胞筛选了 14 种甾体化合物，并鉴定出甾体 A 环 2β-和 D 环 16β-羟化活性。野生型 CYP105D7 能够以低（<10%）转化率催化五种甾体（睾酮、孕酮、4-雄烯-3,17-二酮、醛固酮和皮质酮）的羟化反应。对底物入口和活性位点周围精氨酸残基进行结构导向的定点突变显示，R70A 和 R190A 单突变体以及 R70A/R190A 双突变体显著提高了甾体羟化的转化率。特别是对于睾酮的转化，R70A/R190A 突变体的 / 值增加了 1.35 倍，转化率提高了近 9 倍，具有高度的区域和立体选择性。分子对接分析表明，当 Arg70 和 Arg190 被替换为丙氨酸时，底物进入和结合口袋的体积增加了 1.08 倍，这可能有助于提高甾体的羟化效率。细胞色素 P450 单加氧酶（P450s）能够在温和条件下将氧原子引入非反应性碳氢化合物中，与化学催化剂相比具有显著优势。具有广泛底物特异性和反应多样性的混杂 P450 具有在各个领域应用的巨大潜力，包括合成生物学。研究微生物来源的底物混杂 P450 的功能、分子机制和合理工程化对于发挥这一潜力非常重要。在这里，我们介绍了一种能够催化甾体羟化的微生物底物混杂 P450，即 CYP105D7。活性位点和底物入口中 Arg70 和 Arg190 侧链的缺失导致底物转化率提高了 9 倍。这些发现将支持未来作为生物催化剂应用的 P450 合理和半合理工程化。
