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: 10418782
• 负责人: Kenichi Yokoyama
• 金额: $ 37.6万
• 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/10418782
• 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 inflammation; gut microbiota; human disease; inhibitor; inhibitor therapy; 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.

项目总结/文摘