MycG is a cytochrome P450 that performs two sequential oxidation reactions on the 16-membered ring macrolide M-IV. The enzyme evolved to oxidize M-IV preferentially over M-III and M-VI, which differ only by the presence of methoxy vs free hydroxyl groups on one of the macrolide sugar moieties. We utilized a two-pronged computational approach to study both the chemoselective reactivity and substrate specificity of MycG. Density functional theory computations determined that epoxidation of the substrate hampers its ability to undergo C-H abstraction, primarily due to a loss of hyperconjugation in the transition state. Metadynamics and molecular dynamics simulations revealed a hydrophobic sugar-binding pocket that is responsible for substrate recognition/specificity and was not apparent in crystal structures of the enzyme/substrate complex. Computational results also led to the identification of other interactions between the enzyme and its substrates that had not previously been observed in the cocrystal structures. Site-directed mutagenesis was then employed to test the effects of mutations hypothesized to broaden the substrate scope and alter the product profile of MycG. The results of these experiments validated this complementary effort to engineer MycG variants with improved catalytic activity toward earlier stage mycinamicin substrates.

中文翻译：

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基于计算的机制研究和细胞色素 P450 MycG 选择性氧化 16 元大环内酯类抗生素的工程

MycG 是一种细胞色素 P450，它对 16 元环大环内酯 M-IV 进行两个连续的氧化反应。该酶进化为优先氧化 M-IV，而不是 M-III 和 M-VI，后者的区别仅在于大环内酯糖部分之一上是否存在甲氧基与游离羟基。我们利用双管齐下的计算方法来研究 MycG 的化学选择性反应性和底物特异性。密度泛函理论计算确定，底物的环氧化阻碍了其进行 CH 提取的能力，这主要是由于过渡态中超共轭的损失。元动力学和分子动力学模拟揭示了一个疏水性糖结合袋，它负责底物识别/特异性，并且在酶/底物复合物的晶体结构中并不明显。计算结果还确定了酶与其底物之间的其他相互作用，这些相互作用以前在共晶结构中未观察到。然后采用定点诱变来测试假设的突变的影响，以扩大底物范围并改变 MycG 的产物谱。这些实验的结果验证了这种互补的努力，以设计具有改进的早期霉素底物催化活性的 MycG 变体。