Oxidative Diversification of Steroids by Nature-Inspired Scanning Glycine Mutagenesis of P450BM3 (CYP102A1)
Oxidative Diversification of Steroids by Nature-Inspired Scanning Glycine Mutagenesis of P450BM3 (CYP102A1)
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DOI：
10.1021/acscatal.0c02077
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发表时间：
2020-08-07
期刊：
影响因子：
12.9
通讯作者：
Wong, Luet L.
中科院分区：
文献类型：
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作者：
Chen, Wenyu;Fisher, Matthew J.;Wong, Luet L.
甾体化合物是一些最常用的处方药，被用于治疗各种疾病，包括炎症、心脏病和癌症。功能化类固醇的合成方法对于生成用于药物筛选和开发的类固醇药物具有重要意义。然而，化学激活是具有挑战性的，因为惰性，脂肪族碳氢键在类固醇的优势。在这里，我们报道了来自巨型芽孢杆菌的稳定、高活性的细菌细胞色素P450酶P450BM3 （CYP102A1）的工程，用于雄烯二酮（AD）、脱氢表雄酮（DHEA）和睾酮（TST）的单羟基化和二羟基化。为了设计改变的类固醇结合方向，我们比较了野生型P450BM3与类固醇c19 -去甲基酶CYP19A1的结构，在其活性位点与AD结合，并确定了P450BM3中阻断这种结合方向的I螺旋和β 4链区域。扫描这两个区域的11个残基的甘氨酸诱变导致P450BM3未报道的类固醇氧化产物。在第二轮诱变中结合这些甘氨酸突变导致了一个小的P450BM3变异体库，能够被细菌P450酶在最广泛的位置（C1, C2, C6, C7， C15和C16）选择性（高达97%）氧化AD， DHEA和TST。这些类固醇与选择性P450BM3变异体的分子动力学模拟结构的计算对接表明，甘氨酸突变在实现与野生型不同的结合方向方面发挥了关键作用，包括与CYP19A1中的AD非常相似的突变，而其他突变则微调了产物的选择性。这种从自然界获取灵感设计突变的方法可以应用于其他底物和酶，用于合成天然产物及其衍生物。
Steroidal compounds are some of the most prescribed medicines, being indicated for the treatment of a variety of conditions including inflammation, heart disease, and cancer. Synthetic approaches to functionalized steroids are important for generating steroidal agents for drug screening and development. However, chemical activation is challenging because of the predominance of inert, aliphatic C-H bonds in steroids. Here, we report the engineering of the stable, highly active bacterial cytochrome P450 enzyme P450BM3 (CYP102A1) from Bacillus megaterium for the mono- and dihydroxylation of androstenedione (AD), dehydroepiandrosterone (DHEA), and testosterone (TST). In order to design altered steroid binding orientations, we compared the structure of wild type P450BM3 with the steroid C19-demethylase CYP19A1 with AD bound within its active site and identified regions of the I helix and the beta 4 strand that blocked this binding orientation in P450BM3. Scanning glycine mutagenesis across 11 residues in these two regions led to steroid oxidation products not previously reported for P450BM3. Combining these glycine mutations in a second round of mutagenesis led to a small library of P450BM3 variants capable of selective (up to 97%) oxidation of AD, DHEA, and TST at the widest range of positions (C1, C2, C6, C7, C15, and C16) by a bacterial P450 enzyme. Computational docking of these steroids into molecular dynamics simulated structures of selective P450BM3 variants suggested crucial roles of glycine mutations in enabling different binding orientations from the wild type, including one that closely resembled that of AD in CYP19A1, while other mutations fine-tuned the product selectivity. This approach of designing mutations by taking inspiration from nature can be applied to other substrates and enzymes for the synthesis of natural products and their derivatives.