Catalysis and biogenesis in methylamine dehydrogenase

描述(申请人提供)：甲胺脱氢酶(MADH)是一种古老的细菌代谢酶，在有氧前世界中进化而来。它的厌氧起源被证明是由它催化的高度进化的多步骤化学，以及它在催化过程中使用辅助氧化还原蛋白质伙伴，而不是分子氧来移除电子。尽管是分化进化的产物，但该酶催化的还原化学与含铜胺氧化酶家族相似，人类的相应家族与糖尿病晚期并发症和充血性心脏病的血管变化有关。MADH含有一种新的有机辅因子--色氨酸-色氨酸--色氨酸二酚(TTQ)，对其生物发生的研究将为辅因子如何进化以扩展酶可以控制的化学成分提供新的见解：例如，有助于开发新的工业催化剂的信息。利用单晶可见光谱、X射线结晶学和冷冻捕集的新组合，该项目将探索蛋白质如何通过捕获含有晶体的催化中间体来控制其复杂的多步骤反应，并确定它们的结构。协同突变/结构研究将用于探索活性位点残基在催化、底物结合和TTQ生物发生中的作用。通过研究这种高度发达的代谢系统，这是一种古老的厌氧氧化还原化学物质的一部分，至今仍存在于线粒体和叶绿体中；我们可以揭示利用能量和物质的基本过程，这些物质进化出了地球上复杂的生命。

DESCRIPTION (provided by applicant): Methylamine dehydrogenase (MADH) is an ancient bacterial metabolic enzyme that evolved in a pre-aerobic world. Its anaerobic origins are attested to by the highly evolved multistep chemistry it catalyzes, and its use of ancillary redox protein partners, rather than molecular oxygen, to remove electrons during catalysis. Although a product of divergent evolution, the reductive chemistry catalyzed by the enzyme is analogous to that of the copper-containing amine oxidase family, whose human counterparts are linked to late-diabetic complications and vascular changes in congestive heart disease. MADH contains a novel organic cofactor, tryptophan tryptophylquinone (TTQ), and study of its biogenesis will give new insight into how cofactors have evolved to extend the palette of chemistries enzymes can control: information that could aid in the development of new industrial catalysts, for example. Using a novel combination of single crystal visible spectroscopy, X-ray crystallography and freeze-trapping, this project will probe how the protein controls its complex multistep reaction at atomic resolution by trapping catalytic intermediates in Paracoccus denitrificans MADH (PD-MADH) containing crystals, and determining their structures. Collaborative mutagenesis/structural studies will be used to probe the roles of active site residues in catalysis, substrate binding and TTQ biogenesis. By studying this highly developed metabolic system, a part of the ancient anaerobic redox chemistries still found in mitochondria and chloroplasts today; we can shed light on the fundamental processes of harnessing energy and materials that evolved complex life on earth.