Biosynthesis of Amino Acid Derived Quinone Cofactors

描述(申请人提供)：甲胺脱氢酶(MADH)是一种代谢酶，发现于甲基营养/自养细菌中，含有一种苯醌辅因子色氨酸色氨酸(TTQ)，来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，我们已经开始鉴定其中一种蛋白质，MAUG。它是一种非常不寻常的双血红素酶，负责完成TTQ的合成。MAUG的天然底物(前MADH)是MADH的119 kDa蛋白前体，带有部分形成的辅因子。MAUG催化六电子氧化完成TTQ的生物合成，使用分子氧或过氧化氢作为第二底物。催化反应涉及一种史无前例的高价二价血红素中间体，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。母体资助(GM066569)的具体目标2是解决MAUG与前MADH形成的络合物的晶体结构。这已经达到了2.1°的分辨率，并且这些晶体可以在不损失衍射的情况下支持催化周转来形成TTQ。MAUG/PREMADH结构揭示了意想不到的特征，此次竞争性修订的目标建立在这个新的知识平台上。利用定点突变、X射线结晶学、动力学表征、光谱学和质谱学，这一竞争性修订将使我们能够迅速发现以前未知的高价铁中间体的氧活化、蛋白质间长程电子和自由基转移以及蛋白质中特定氨基酸残基的氧化修饰机制。 与公共健康相关：MAUG顺序与过氧化物酶相关，在低氧条件下(氧化应激)可使过氧化氢解毒，但通常也能激活分子氧，并使用与人类细胞色素P450酶相当的催化中间体，后者是药物代谢、致癌物质激活、类固醇、脂溶维生素和脂肪酸等重要生理分子的生物合成以及杀虫剂和除草剂降解的主要参与者。

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.