Selective oxidation of carbolide C-H bonds by an engineered macrolide P450 mono-oxygenase
Selective oxidation of carbolide C-H bonds by an engineered macrolide P450 mono-oxygenase
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DOI：
10.1073/pnas.0907203106
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发表时间：
2009-11-03
影响因子：
11.1
通讯作者：
Sherman, David H.
中科院分区：
文献类型：
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作者：
Li, Shengying;Chaulagain, Mani Raj;Sherman, David H.
未活化的 C-H 键的区域选择性和立体选择性氧化仍然是有机化学中的一个核心挑战。人们投入了大量精力来识别过渡金属配合物、生物催化剂或简化的模拟物，但取得的成功有限。细胞色素 P450 单加氧酶参与多种类型的区域选择性和立体选择性氧化，是应对这一挑战的有前途的生物催化剂。如果这类酶的底物谱可以拓宽、选择性可控并且在没有昂贵的异源氧化还原伙伴的情况下催化反应，那么这类酶的应用就特别重要。在这项研究中，我们设计了一种大环内酯类生物合成P450单加氧酶PikC (PikC(D50N)-RhFRED)，它具有显着的底物灵活性，与野生型酶相比活性显着提高，并且能够自给自足。通过迄今为止尚未充分探索的“底物工程”策略，利用其独特的去糖胺锚定功能，我们证明了PikC能够以区域选择性方式羟基化一系列通过缩醛键（称为“碳内酯”）与去糖胺糖苷连接的碳环。对许多高分辨率酶-底物共晶结构的补充分析为了解氨基糖衍生的锚定基团控制反应位点选择性的功能提供了重要的见解。此外，其中一些碳内酯系统的意想不到的生物活性揭示了它们作为先前未记录的抗生素类别的潜力。
Regio- and stereoselective oxidation of an unactivated C-H bond remains a central challenge in organic chemistry. Considerable effort has been devoted to identifying transition metal complexes, biological catalysts, or simplified mimics, but limited success has been achieved. Cytochrome P450 mono-oxygenases are involved in diverse types of regio- and stereoselective oxidations, and represent a promising biocatalyst to address this challenge. The application of this class of enzymes is particularly significant if their substrate spectra can be broadened, selectivity controlled, and reactions catalyzed in the absence of expensive heterologous redox partners. In this study, we engineered a macrolide biosynthetic P450 mono-oxygenase PikC (PikC(D50N)-RhFRED) with remarkable substrate flexibility, significantly increased activity compared to wild-type enzyme, and self-sufficiency. By harnessing its unique desosamine-anchoring functionality via a heretofore under-explored "substrate engineering'' strategy, we demonstrated the ability of PikC to hydroxylate a series of carbocyclic rings linked to the desosamine glycoside via an acetal linkage (referred to as "carbolides'') in a regioselective manner. Complementary analysis of a number of high-resolution enzyme-substrate cocrystal structures provided significant insights into the function of the aminosugar-derived anchoring group for control of reaction site selectivity. Moreover, unexpected biological activity of a select number of these carbolide systems revealed their potential as a previously unrecorded class of antibiotics.