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: 10242931
• 负责人: Kenichi Yokoyama
• 金额: $ 37.46万
• 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/10242931
• 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.

项目总结/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，它是必不可少的， 正常的大脑发育和莫科生物合成基因的突变导致致命的，目前无法治愈的 Moco缺乏症（MoCD）。在致病菌中，Moco对于它们在缺氧和缺氧环境下的生长是必不可少的。 营养限制环境，因此是病原体在哺乳动物宿主中持续存在的必要条件。慢性 细菌感染对许多抗生素具有抗药性，并导致急性症状复发。但 开发针对MoCD的治疗剂或靶向病原菌中Moco生物合成的抗生素， 由于我们对Moco生物合成机制的了解有限，目前很难进行。远景目标 该项目的目的是提供致病菌中Moco生物合成的机制理解， 在人类身上。当前申请的重点是两种酶（MoaA和MoaC）的作用机制 用于从鸟嘌呤5 '-三磷酸（GTP）形成Moco的吡喃蝶呤结构。虽然催化剂 MoaA和MoaC的功能在20多年来一直不明确，我们最近证明MoaA 催化GTP转化为3 '，8-cyclo-dihydro-GTP（3'，8-cH 2GTP），而MoaC催化 将3 '，8-cH 2GTP转化为cPMP。在本申请中，我们将研究MoaA的催化机制 和MoaC在人类和细菌通过以下三个目的。在目标1中，4Fe-4S的氧化还原功能 在MoaA集群将在休息状态和营业额进行调查，以解决一个关键的 自由基SAM酶机制的未解之谜在目标2中，MoaA的C末端尾的功能是 通过核磁共振、X射线衍射、核磁共振、X射线衍射等手段， 晶体学和使用细菌和人类酶的生物化学测定。在目标3中，我们将测试共价键 和非共价机制MoaC催化和调查的一般性机制为基础的抑制 细菌和人类酶的混合物。这项研究具有重要意义，因为它将提供 深入了解Moco骨干的形成和Moco未来发展的科学基础 生物合成抑制剂和治疗MoCD的新疗法。