Enzymology of cofactor and amino acid metabolism in anaerobic bacteria

在过去的十年里，我的nserc资助的研究项目提供了对辅酶介导的机制的重要见解，例如辅酶- b12自由基化学，黄素介导的电子转移和单氧作用以及PLP催化的ß-置换反应。在接下来的五年里，我将把我的项目集中在梭杆菌门中血红素、B12和阳离子氨基酸代谢的酶和辅助蛋白上。厌氧赖氨酸发酵已被证明可以控制由口腔细菌核梭杆菌介导的种间相互作用，以及兽医病原体坏死梭杆菌的增殖和毒力。我们的第一个目标是表征赖氨酸发酵途径中剩余的两个未注释的基因产物。我假设这些蛋白质作为新的金属伴侣，重新激活辅酶B12酶，催化发酵途径的第二步。这条调查线建立在我现有的研究力量和NSERC资助的研究项目上，研究由辅酶B12氨基转化酶催化的自由基介导的反应。它还旨在扩大我们对辅酶B12酶对抗代谢昂贵的B12辅助因子的非定常损伤的机制的认识。我们研究计划的第二个主要目标是阐明从核仁梭菌中提取的鸟氨酸脱羧酶/精氨酸酶的结构、功能和生理作用。我假设这种酶通过产生多胺，腐烂来支持生物膜的生长。我们的目标是确定酶的结构，并开发酶及其活性变体的完整功能表征。我们还旨在通过基因破坏和互补实验确定该蛋白的生理作用。这条探究线将拓宽我们的研究专长，同时扩大我们对这种酶在生物膜发展中的作用的认识。我们的第三个目标是在功能和结构上表征一种具有结合血红素能力的新型黄氧还蛋白。基于其基因组位置，我假设它在自由基介导的铁获取途径中既充当还原剂又充当血红素伴侣。这个研究项目也建立在我在黄素介导的电子转移和自由基介导的酶促反应方面的专业知识之上。这项研究还将深入了解一些常见的厌氧菌是如何在口腔中获取铁的，目前对这一点知之甚少。参与该研究项目的高素质人才将有机会在跨越生物物理学、生物化学、遗传学、微生物学和分子生物学等学科的方法上发展技术专长。他们还将有机会参与合作研究和批判性分析，为他们在学术界、工业界和政府的职业生涯做好准备。

Over the past decade, my NSERC-funded research program has provided critical insight into mechanisms mediated by coenzyme containing enzymes, for example coenzyme-B12 radical chemistry, flavin-mediated electron transfer and monooxygenations and ß-replacement reactions catalyzed by PLP. Over the next five years, I will focus my program on enzymes and accessory proteins that function in heme, B12 and cationic amino acid metabolism in the phyla of Fusobacteria. Anaerobic lysine fermentation has been shown to control interspecies interactions mediated by the oral bacterium Fusobacterium nucleatum as well as the proliferation and virulence of the veterinary pathogen, Fusobacterium necrophorum. Our first objective is to characterize the two remaining unannotated gene products in the lysine fermentation pathway. I hypothesize that these proteins serve as novel metallochaperones that function to reactivate a coenzyme B12 enzyme, which catalyzes the second step in the fermentation pathway. This line of inquiry builds upon my existing research strength and NSERC- funded research program in radical-mediated reactions catalyzed by coenzyme B12 aminomutases. It also aims to expand our knowledge of the mechanisms by which coenzyme B12-enzymes contend with adventitious damage to the metabolically expensive B12 cofactor. The second main objective of our research program is to elucidate the structure, function and physiological role of an ornithine decarboxylase/arginase from F. nucleatum. I hypothesize that this enzyme supports biofilm growth through production of the polyamine, putrescence. We aim to determine the structure of the enzyme and develop a complete functional characterization of the enzyme and its active variants. We also aim to determine the physiological role of the protein through gene disruption and complementation experiments. This line of inquiry will broaden our research expertise while expanding our knowledge of the role of this enzyme in development of biofilm. Our third objective is to functionally and structurally characterize a novel flavodoxin that has the capacity to bind heme. Based on its genomic location, I hypothesize that serves as both a reductant and heme chaperone in a radical-mediated iron acquisition pathway. This research project also builds upon my expertise in flavin-mediated electron transfer and radical-mediated enzymatic reactions. The research will also provide insight into how some prevalent anaerobic bacteria acquire iron in the oral cavity, for which little is currently known. Highly qualified personnel engaged in this research program will have the opportunity develop technical expertise in methods that span the disciplines of biophysics, biochemistry, genetics, microbiology and molecular biology. They will also have the opportunity to engage in collaborative research and critical analysis, preparing them for fulfilling careers in academia, industry and government.