Biosynthesis of Amino Acid Derived Quinone Cofactors

描述（由申请人提供）：甲胺脱氢酶（MADH）是一种在甲基营养/自养细菌中发现的代谢酶，含有醌辅因子色氨酸甲萘醌（TTQ），来源于蛋白质中两个Trp残基的翻译后修饰。MADH的成熟涉及至少4种其他蛋白质，我们已经开始表征这些蛋白质之一，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催化六电子氧化以完成TTQ生物合成，使用分子氧或过氧化氢作为第二底物。催化反应涉及前所未有的高价二血红素中间体，它异常稳定。本申请是对“NOT-OD-09-058：NIH宣布恢复法案资金可用于竞争性修订申请”的回应。“母基金（GM 066569）的具体目标2是解决MauG与preMADH复合的晶体结构。这已经达到了2.1英寸的分辨率，这些晶体可以支持催化周转形成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的是建立在这个新的知识平台上。使用定点诱变，X-射线晶体学，动力学表征，光谱和质谱这种竞争性的修订将使我们能够迅速作出关于氧活化的一个以前未知的高价铁中间体，长距离蛋白质间电子和自由基转移和机制的氧化修饰的蛋白质内的特定氨基酸残基的基本发现。 公共卫生相关性：MauG依次与在缺氧条件下解毒H2 O2的过氧化物酶相关（氧化应激），但不寻常的是也可以激活分子氧，并使用与人细胞色素P450酶相当的催化中间体，细胞色素P450酶是药物代谢、致癌物活化、生理学上重要的分子如类固醇、脂溶性维生素和脂肪酸的生物合成的主要参与者，以及杀虫剂和除草剂的降解。

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.