Mechanism of carbon skeleton formation in molybdenum cofactor biosynthesis

钼辅因子生物合成中碳骨架形成机制

• 批准号: 10058693
• 负责人: Kenichi Yokoyama
• 金额: $ 45.78万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10058693
• 关键词: Active Sites; Address; Affect; Anabolism; Antibiotics; Bacteria; Bacterial Infections; Biochemical; Biological Assay; Brain; C-terminal; Catalysis; Cessation of life; Childhood; Chronic; Coenzymes; Combined molybdoflavoprotein enzyme deficiency; Communicable Diseases; Complex; Development; Disease; Electron Nuclear Double Resonance; Enterobacteriaceae; Environment; Enzymes; Family; Foundations; Funding; Future; Genes; Goals; Growth; Guanine; Guanosine; Human; Hypoxia; Individual; Inherited; Knowledge; Mutation; Nutrient; Organism; Oxidation-Reduction; Pathway interactions; Patients; Peptides; Periodicity; Pharmacology; Proteins; Public Health; Reaction; Recurrence; Research; Resistance; Rest; Severities; Site; Structure; Tail; Testing; Vertebral column; X-Ray Crystallography; acute symptom; base; biophysical techniques; carbon skeleton; chronic infection; cofactor; combat; electron donor; enzyme mechanism; gut microbiota; human disease; inflammatory disease of the intestine; inhibitor/antagonist; insight; loss of function mutation; molybdenum cofactor; novel therapeutics; pathogen; pathogenic bacteria; pyranopterin; therapeutic development; tripolyphosphate

Project Summary/Abstract Molybdenum cofactor (Moco) is a redox cofactor found in almost all organisms. In humans, it is essential for normal brain development, and mutations in Moco biosynthetic genes cause the fatal and currently incurable disease, Moco deficiency (MoCD). In pathogenic bacteria, Moco is essential for their growth under hypoxic and nutrient limiting environments, and therefore essential for pathogen persistence in mammalian hosts. Chronic bacterial infections are resistant to many antibiotics and cause the recurrence of acute symptoms. However, the development of therapeutics against MoCD or antibiotics targeting Moco biosynthesis in pathogenic bacteria are currently difficult due to our limited understanding of the mechanism of Moco biosynthesis. The long-term goal of this project is to provide a mechanistic understanding of Moco biosynthesis in pathogenic bacteria as well as in humans. The focus of the current application is the mechanism of two enzymes (MoaA and MoaC) responsible for the formation of the pyranopterin structure of Moco from guanine 5'-triphosphate (GTP). While the catalytic functions of MoaA and MoaC had remained ambiguous for >20 years, we recently demonstrated that MoaA catalyzes the transformation of GTP into 3',8-cyclo-dihydro-GTP (3',8-cH2GTP), while MoaC catalyzes the conversion of 3',8-cH2GTP to cPMP. In this application, we will investigate the catalytic mechanisms of MoaA and MoaC in both humans and bacteria through the following three Aims. In Aim 1, the redox function of 4Fe-4S clusters in MoaA will be investigated both in the resting state and during turnover to address one of the key unsolved mysteries of the radical SAM enzyme mechanisms. In Aim 2, the function of the C-terminal tail of MoaA and the mechanism of peptide rescue of MoCD-causing mutations will be investigated through NMR, X-ray crystallography and biochemical assays using bacterial and human enzymes. In Aim 3, we will test a covalent and non-covalent mechanisms for MoaC catalysis and investigate the generality of mechanism-based inhibition of bacterial and human enzymes. The proposed research is significant because it will provide mechanistic insights into the formation of the Moco backbone and the scientific basis for future development of Moco biosynthesis inhibitors and novel therapeutics to treat MoCD.

项目摘要/摘要 钼辅因子（MOCO）是几乎所有生物体中发现的氧化还原辅因子。在人类中，这对于 正常的大脑发育和Moco生物合成基因的突变会导致致命且目前无法治愈 疾病，MOCO缺乏症（MOCD）。在致病性细菌中，Moco对于低氧和低氧的生长至关重要 营养限制环境，因此对于哺乳动物宿主的病原体持久性至关重要。慢性的 细菌感染对许多抗生素具有抗性，并引起急性症状的复发。但是， 针对靶向MOCO生物合成的致病细菌中的MOCD或抗生素的疗法开发是 由于我们对MoCO生物合成机制的理解有限，目前很难。长期目标 这个项目的是对致病细菌中的Moco生物合成的机械理解 在人类中。当前应用的重点是两种酶（MOAA和MOAC）的机制 为了形成来自鸟嘌呤5'-三磷酸盐（GTP）的Moco的吡喃翅目结构。而催化 MOAA和MOAC的功能一直模棱两可超过20年，我们最近证明了MOAA 催化GTP转化为3'，8-Cyclo-dihydro-GTP（3'，8-CH2GTP），而MOAC则催化 将3'，8-CH2GTP转换为CPMP。在此应用中，我们将研究MOAA的催化机制 通过以下三个目标，人类和细菌的MOAC和细菌。在AIM 1中，4FE-4S的氧化还原功能 MOAA中的集群将在静止状态和营业额期间进行调查，以解决关键之一 激进的SAM酶机制的未解决的奥秘。在AIM 2中，MOAA的C末端尾巴的功能 并将通过NMR，X射线研究肽挽救MOCD引起的突变的机制 使用细菌和人类酶的晶体学和生化测定。在AIM 3中，我们将测试共价 MOAC催化的非共价机制，并研究基于机制的抑制的一般性 细菌和人类酶。拟议的研究很重要，因为它将提供机械 对Moco主链的形成和Moco未来发展的科学基础的见解 生物合成抑制剂和新型治疗MOCD的疗法。