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，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，使用三个摩尔分子氧或过氧化氢作为第二底物。MAUG的两个血红素作为一个单一的氧化还原单元，催化反应涉及一种史无前例的高价二价血红素中间体，这种中间体异常稳定。高价物种是相当于FeV的物种，由一个FeIV=O血红素组成，另一个处于FeIV氧化态。在之前的资助期间，我们已经解决了MAUG与前MADH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的结构，以及来自合作实验室的其他最新数据，都充满了惊喜。结构表明，在催化过程中没有主要的结构重排，该体系中发生了长程的蛋白质间电子转移和自由基转移。在催化过程中，还会产生一种非常稳定的MADH蛋白质自由基。现在，我们希望将结晶学研究，辅以质谱学和单晶光谱学，超越前一个资助期的预期，并在以前未知的高价铁中间体的氧活化、蛋白质中特定氨基酸残基的氧化修饰机制以及高活性氧物种(ROS)和自由基的稳定方面取得具体发现。从根本上说，这些研究将使人们对代谢物中心的蛋白质间电子转移，以及与许多疾病状态相关的自由基和ROS对氧化损伤的控制和结果有更深入的了解。因此，对蛋白质基质中的电子、自由基和ROS进行保护性控制是人类健康的关键组成部分。

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, 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 three moles of either molecular oxygen or hydrogen peroxide as the second substrate. The two hemes of MauG act as a single redox unit, and the catalytic reaction involves an unprecedented high-valent di-heme intermediate that is unusually stable. The high-valent species is an FeV equivalent consisting of one FeIV=O heme, with the other in an FeIV oxidation state. During the previous funding period we have solved 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, along with other recent data from collaborating labs, has been full of surprises. The structure indicates there are no major structural rearrangements during catalysis and that long-range inter-protein electron and radical transfer occurs in this system. During catalysis a very stable MADH protein radical is also produced. Now we wish to move the crystallographic studies, complemented by mass spectrometry and single crystal spectroscopies, beyond what was envisioned during the previous funding period, and make specific discoveries about oxygen activation by a previously unknown high-valent iron intermediate, mechanisms of oxidative modification to specific amino acid residues within a protein, and stabilization of highly reactive oxygen species (ROS) and radicals. Fundamentally these studies will give molecular insight into inter-protein electron transfer central to metabolis, and the control and outcomes of oxidative damage by radicals and ROS that are associated with many disease states. Thus protective control of electrons, radicals and ROS within the protein matrix is a key component of human health.