Studies of metal-containing and metal-less EutT adenosyltransferases

项目总结/摘要 了解病原体代谢需求的生理背景为以下方面提供了框架： 有针对性地开发抗菌药物。在肠道沙门氏菌的情况下，乙醇胺的呼吸在 肠道环境提供了优于肠道微生物的适应性优势。乙醇胺 辅酶B12是一种辅酶B12依赖性途径，发生在一个称为 乙醇胺（Eut）代谢体。乙醇胺通过乙醇胺氨裂解酶（EAL）脱氨基， 形成乙醛，最终以乙酰辅酶A的形式进入中枢代谢。EutA重新激活非活动EAL 通过以ATP为代价去除功能失调的辅酶B12。需要这些反应来触发 乙醇胺催化剂的初始步骤，从而了解这些过程所需的相互作用， Eut代谢体的背景将提供靶向破坏代谢的机会， 通路有几个知识空白需要填补，这样我们就可以提高我们的理解， 乙醇胺催化剂在这种人类病原体中。第一，腺苷转移酶的催化机制 将维生素B12转化为辅酶B12的EutT酶未知，因此将进行研究。而S. 肠EutT是一种金属蛋白，在一些其他病原体中的EutT同源物（例如，单核细胞增生李斯特菌， 破伤风梭菌）在没有金属的情况下发挥作用。将研究含金属和无金属的Eut酶， 它们的催化机制进行了比较，以阐明金属中心的作用。二是不理解 辅酶B12是如何从EutT输送到EAL的。初步证据强烈表明，EutA介导 EutT、EutA和EAL可以形成复合物。多学科方法（晶体学， 光谱学、分子生物学、生物化学、体内遗传学和生理学）将用于解决这一问题 复杂的问题总的来说，这项工作将促进我们对细胞如何合成和传递的理解。 必需的辅酶与使用它们的酶（在这种情况下是在细胞隔室内）。

Project Summary/Abstract Understanding the physiological context of the metabolic needs of a pathogen provides a framework for targeted development of antimicrobials. In the case of Salmonella enterica, respiration of ethanolamine in the intestinal environment provides a fitness advantage over commensal microorganisms. Ethanolamine catabolism is a coenzyme B12-dependent pathway that occurs inside a proteinaceous compartment called the ethanolamine (Eut) metabolosome. Ethanolamine is deaminated by ethanolamine ammonia lyase (EAL) to form acetaldehyde, which eventually enters central metabolism as acetyl-CoA. EutA reactivates inactive EAL by removing dysfunctional coenzyme B12 at the expense of ATP. These reactions are needed to trigger the initial step of ethanolamine catabolism hence understanding the interactions required for these processes in the context of the Eut metabolosome would provide opportunities for targeted disruption of the metabolic pathway. There are several gaps of knowledge that need to be filled in so we can improve our understanding f ethanolamine catabolism in this human pathogen. First, the mechanism of catalysis of the adenosyltransferase EutT enzyme that converts vitamin B12 to coenzyme B12 is unknown, thus will be investigated. While the S. enterica EutT is a metalloprotein, EutT homologues in some other pathogens (e.g., Listeria monocytogenes, Clostridum tetani) function without a metal. Metal containing and metal-less Eut enzyme will be studied, and their mechanisms of catalysis compared to elucidate the role of the metal center. Second, it is not understood how coenzyme B12 is delivered to EAL from EutT. Preliminary evidence strongly suggests that EutA mediates the delivery, and that EutT, EutA and EAL may form a complex. A multidisciplinary approach (crystallography, spectroscopy, molecular biology, biochemistry, in vivo genetics, and physiology) will be used to address this complex problem. Collectively, this work will advance our understanding of how cells synthesize and deliver essential coenzymes to the enzymes that use them, in this case inside a cellular compartment.