Joint BioEnergy Institute, Emeryville, CA 94608.
Biomass Science and Conversion Technology Department, Sandia National Laboratories, Livermore, CA 94550.
Proc Natl Acad Sci U S A. 2017 Apr 18;114(16):E3205-E3214. doi: 10.1073/pnas.1619263114. Epub 2017 Apr 3.
Some strains of soil and marine bacteria have evolved intricate metabolic pathways for using environmentally derived aromatics as a carbon source. Many of these metabolic pathways go through intermediates such as vanillate, 3--methylgallate, and syringate. Demethylation of these compounds is essential for downstream aryl modification, ring opening, and subsequent assimilation of these compounds into the tricarboxylic acid (TCA) cycle, and, correspondingly, there are a variety of associated aryl demethylase systems that vary in complexity. Intriguingly, only a basic understanding of the least complex system, the tetrahydrofolate-dependent aryl demethylase LigM from , a bacterial strain that metabolizes lignin-derived aromatics, was previously available. LigM-catalyzed demethylation enables further modification and ring opening of the single-ring aromatics vanillate and 3--methylgallate, which are common byproducts of biofuel production. Here, we characterize aryl -demethylation by LigM and report its 1.81-Å crystal structure, revealing a unique demethylase fold and a canonical folate-binding domain. Structural homology and geometry optimization calculations enabled the identification of LigM's tetrahydrofolate-binding site and protein-folate interactions. Computationally guided mutagenesis and kinetic analyses allowed the identification of the enzyme's aryl-binding site location and determination of its unique, catalytic tyrosine-dependent reaction mechanism. This work defines LigM as a distinct demethylase, both structurally and functionally, and provides insight into demethylation and its reaction requirements. These results afford the mechanistic details required for efficient utilization of LigM as a tool for aryl -demethylation and as a component of synthetic biology efforts to valorize previously underused aromatic compounds.
一些土壤和海洋细菌已经进化出复杂的代谢途径，可将环境衍生的芳烃作为碳源。这些代谢途径中的许多途径都经过香草酸盐、3--甲基没食子酸和丁香酸盐等中间体。这些化合物的脱甲基化对于下游芳基修饰、开环以及随后将这些化合物同化到三羧酸 (TCA) 循环中至关重要，因此存在各种相关的芳基脱甲基酶系统，其复杂性各不相同。有趣的是，以前仅对最简单的系统——来自代谢木质素衍生芳烃的细菌菌株 的四氢叶酸依赖性芳基脱甲基酶 LigM 有基本的了解。LigM 催化的脱甲基作用使单环芳烃香草酸盐和 3--甲基没食子酸能够进一步修饰和开环，这两种物质都是生物燃料生产的常见副产物。在这里，我们描述了 LigM 的芳基脱甲基作用，并报告了其 1.81-Å 晶体结构，揭示了一种独特的脱甲基酶折叠和典型的叶酸结合域。结构同源性和几何优化计算使我们能够确定 LigM 的四氢叶酸结合位点和蛋白-叶酸相互作用。计算指导的诱变和动力学分析使我们能够确定酶的芳基结合位点位置，并确定其独特的、催化酪氨酸依赖性反应机制。这项工作将 LigM 定义为在结构和功能上都不同的脱甲基酶，并深入了解脱甲基作用及其反应要求。这些结果提供了有效利用 LigM 作为芳基脱甲基工具以及作为合成生物学努力利用以前未充分利用的芳香族化合物的组件所需的机制细节。
