Analysis of Metabolic Capabilities of Prokaryotic Cells

项目主任/首席调查员(最后、第一、中间)：埃斯卡兰特，豪尔赫·C。 项目摘要/摘要 这项米拉建议将原核生物代谢和生理两个领域结合在一起，PI小组 对……做出了广泛贡献。第一种是结构复杂的辅酶B12(CoB12)的组装，以及 第二个是赖氨酸乙酰化对蛋白质功能的调节，以响应代谢应激。 PI的团队之前在这些领域的工作已经导致了新的酶和途径的发现， 并建立了适用于生命所有领域的细胞的基本生理学范式。 关于复杂的辅酶B12是如何形成的，我们已经了解了很多，然而，我们的知识空白 关于它的组装仍然存在。尽管剩余的差距很难解决，但最近在我们的 小组已经开发了工具来解决这些问题，并促进了我们对 CoB12生物合成在具有社会重要性的微生物中的生理整合。我们会调查的 在人类病原体中，CoB12的低配体碱基是如何合成并激活其核苷的 维生素B12在几种革兰氏阳性病原体中被转化为CoB12，为什么该途径的最后几步 在迄今已知的所有CoB12生产者中，都发生在细胞膜上。大部分拟议的工作都将完成 肠道沙门氏菌，因为我们对这种细菌中CoB12的生物合成有深入的了解，而且 复杂的遗传系统可用于体内工作。我们还将使用沙门氏菌来建立功能 在其他细菌和古菌中具有异源的、推定的CoB12生物合成功能。 我们将继续研究赖氨酸乙酰化在控制代谢应激中的作用。赖氨酸 乙酰化是一种翻译后修饰，与人类健康和生物技术密切相关。这个 这一调节机制对人类细胞衰老和癌症、神经退行性疾病、糖尿病、 肥胖、抗菌素耐药性、微生物发病机制和其他与社会相关的研究领域 强调需要继续推进这一研究领域。关于赖氨酸的基本问题 乙酰化反应仍未得到回答。拟议的工作将研究原核生物sirtuin的新角色(S) 脱乙酰酶在原核生理学中的作用，并将继续阐明脱乙酰酶的功能和生理作用 革兰氏阴性和革兰氏阳性人类病原体中的乙酰转移酶。我们的调查结果来自 用原核模型生物进行的实验将告知该系统如何在更高的形式下工作 生活的一部分。 一种强大、创新的方法组合，包括过渡金属光谱学、结构生物学 (结晶学)、生物化学、分子生物学、体内遗传学、生理学、单分子生物物理学、 在执行拟议工作期间，将进行全系统分析。我们将合作 与光谱学、结晶学、分子生物物理学和膜生物学领域的专家一起 为假设和工作模型提供全面、严格的测试。 OMB编号0925-0001/0002(03/16修订版批准至2018年10月31日)页面续格式页面

Program Director/Principal Investigator (Last, First, Middle): Escalante, Jorge C. PROJECT SUMMARY/ABSTRACT This MIRA proposal brings together two fields of prokaryotic metabolism and physiology that the PI’s group has contributed extensively to. The first is the assembly of the structurally complex coenzyme B12 (CoB12), and the second one is the regulation of protein function by lysine acetylation in response to metabolic stress. Previous work by the PI’s group in these areas has resulted in the discovery of new enzymes and pathways, and has established fundamental physiologic paradigms that apply to cells of all domains of life. We have learned a great deal about how the complex coenzyme B12 is made, and yet, gaps in our knowledge about its assembly remain. Although the remaining gaps are challenging to solve, recent breakthroughs in our group have generated the tools to address these questions and advance our understanding of the physiological integration of CoB12 biosynthesis in microorganisms of societal importance. We will investigate how the lower ligand base of CoB12 is synthesized and activated to its riboside in human pathogens, how vitamin B12 is converted to CoB12 in several Gram-positive pathogens, and why the last steps of the pathway occur at the cell membrane in all CoB12 producers known to date. Most of the proposed work will be performed in Salmonella enterica because of our deep knowledge of CoB12 biosynthesis in this bacterium, and the sophisticated genetic system available to do in vivo work. We will also use Salmonella to establish the function of heterologous, putative CoB12 biosynthetic functions in other bacteria and archaea. We will continue to investigate the role of lysine acetylation in the control of metabolic stress. Lysine acetylation is a posttranslational modification of profound relevance to human health and biotechnology. The impact of this regulatory mechanism on human cell aging and cancer, neurodegenerative diseases, diabetes, obesity, antimicrobial resistance, microbial pathogenesis, and other research areas of societal relevance emphasizes the need to continue advancing this field of research. Fundamental questions about lysine acetylation remain unanswered. The proposed work will investigate new role(s) of prokaryotic sirtuin deacetylases in prokaryotic physiology, and will continue to elucidate the functions and physiological roles of acetyltransferases in Gram-negative and Gram-positive human pathogens. Our findings obtained from experiments performed with prokaryotic model organisms will inform how the system may work in higher forms of life. A powerful, innovative combination of approaches, including transition metal spectroscopy, structural biology (crystallography), biochemistry, molecular biology, in vivo genetics, physiology, single-molecule biophysics, and system-wide analyses will be applied during the performance of the proposed work. We will collaborate with experts in the fields of spectroscopy, crystallography, molecular biophysics, and membrane biology to provide comprehensive, rigorous testing of hypotheses and working models. OMB No. 0925-0001/0002 (Rev. 03/16 Approved Through 10/31/2018) Page Continuation Format Page