Biosynthesis of Amino Acid Derived Quinone Cofactors

描述（由申请人提供）：甲胺脱氢酶（MADH）是甲基营养型/自养型细菌中发现的一种代谢酶，含有醌辅因子色氨酸色氨酸醌（TTQ），源自蛋白质中两个色氨酸残基的翻译后修饰。 MADH 的成熟至少涉及 4 种其他蛋白质，我们已经开始表征其中一种蛋白质 MauG。它是一种非常不寻常的二血红素酶，负责完成 TTQ 合成。 MauG (preMADH) 的天然底物是 MADH 的 119 kDa 蛋白质前体，具有部分形成的辅因子。 MauG 使用分子氧或过氧化氢作为第二底物催化六电子氧化来完成 TTQ 生物合成。催化反应涉及前所未有的高价二血红素中间体，该中间体异常稳定。此申请是对“NOT-OD-09-058：NIH 宣布恢复法案基金可用于竞争性修订申请”的回应。母基金 (GM066569) 的具体目标 2 是解析 MauG 与 preMADH 复合物的晶体结构。分辨率已达到 2.1¿，并且这些晶体可以支持催化转换以形成 TTQ，而不会损失衍射。 MauG/preMADH 结构揭示了未预料到的功能，本次竞争性修订的目标是建立在这个新的知识平台上。利用定点诱变、X射线晶体学、动力学表征、光谱学和质谱法，这一竞争性修订将使我们能够快速做出关于以前未知的高价铁中间体的氧活化、长距离蛋白质间电子和自由基转移以及蛋白质内特定氨基酸残基的氧化修饰机制的基本发现。 公共健康相关性：MauG 依次与在缺氧条件下（氧化应激）解毒 H2O2 的过氧化物酶相关，但通常也可以激活分子氧，并使用与人类细胞色素 P450 酶等效的催化中间体，细胞色素 P450 酶是药物代谢、致癌物激活、生理重要分子（如类固醇、脂溶性维生素和脂肪酸）生物合成以及杀虫剂降解的主要参与者和除草剂。

DESCRIPTION (provided by applicant):Methylamine dehydrogenase (MADH), a metabolic enzyme found in methylotrophic/autotrophic bacteria, contains a quinone cofactor, tryptophan tryptophylquinone (TTQ), derived from the post-translational modification of two Trp residues in the protein. The maturation of MADH involves at least 4 other proteins, and we have begun to characterize one of these proteins, MauG. It is a highly unusual di-heme enzyme responsible for the completion of TTQ synthesis. The natural substrate for MauG (preMADH) is a 119-kDa protein precursor of MADH with a partially formed cofactor. MauG catalyzes a six-electron oxidation to complete TTQ biosynthesis, using either molecular oxygen or hydrogen peroxide as the second substrate. The catalytic reaction involves an unprecedented high-valent di-heme intermediate, which is unusually stable. This application is in response to "NOT-OD-09-058: NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications." Specific Aim 2 of the parent grant (GM066569) was to solve the crystal structure of MauG in complex with preMADH. This has been achieved to a resolution of 2.1¿, and these crystals can support catalytic turnover to form TTQ without loss of diffraction. The MauG/preMADH structure has revealed features that were not anticipated, and the aims of this competitive revision are built on this new knowledge platform. Using site-directed mutagenesis, X-ray crystallography, kinetic characterization, spectroscopy and mass spectrometry this competitive revision would enable us to rapidly make fundamental discoveries about oxygen activation by a previously unknown high-valent iron intermediate, long range inter-protein electron and radical transfer and mechanisms of oxidative modification to specific amino acid residues within a protein. PUBLIC HEALTH RELEVANCE: MauG is sequentially related to peroxidases that detoxify H2O2 under hypoxic conditions (oxidative stress), but unusually can also activate molecular oxygen, and uses an equivalent catalytic intermediate to human cytochrome P450 enzymes, the major players in drug metabolism, carcinogen activation, biosynthesis of physiologically important molecules, such as steroids, fat-soluble vitamins and fatty acids, as well as the degradation of insecticides and herbicides.