Catalysis and biogenesis in methylamine dehydrogenase

描述（由申请人提供）：甲胺脱氢酶（MADH）是一种古老的细菌代谢酶，在有氧前世界进化而来。它的厌氧起源是由它催化的高度进化的多步化学反应证明的，它使用辅助氧化还原蛋白伙伴，而不是分子氧，在催化过程中去除电子。虽然这种酶是分化进化的产物，但它催化的还原化学与含铜胺氧化酶家族类似，后者与人类的晚期糖尿病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助因子，色氨酸色氨酸醌（TTQ），对其生物发生的研究将使我们对辅助因子如何进化以扩展酶可以控制的化学调色板有新的认识：例如，这些信息可能有助于开发新的工业催化剂。利用单晶可见光谱、x射线晶体学和冷冻捕获的新组合，该项目将通过在含反硝化副球菌MADH （PD-MADH）的晶体中捕获催化中间体，并确定其结构，探索蛋白质如何在原子分辨率上控制其复杂的多步骤反应。协同诱变/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。通过研究这个高度发达的代谢系统，线粒体和叶绿体中仍然存在古代厌氧氧化还原化学物质的一部分；我们可以阐明利用能源和物质的基本过程，这些过程使地球上的复杂生命得以进化。

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.