Mechanism of carbon skeleton formation in molybdenum cofactor biosynthesis

项目摘要/摘要 钼辅因子(MoCo)是一种几乎存在于所有生物体中的氧化还原辅因子。在人类中，它是必不可少的 大脑正常发育和MOCO生物合成基因突变导致致命的目前无法治愈的 疾病，莫可缺乏(MoCD)。在病原菌中，MOCO对它们在低氧和低氧条件下的生长是必不可少的 营养限制环境，因此对病原体在哺乳动物宿主中的持久性是必不可少的。慢性 细菌感染对许多抗生素具有抗药性，并会导致急性症状的复发。然而， 针对病原菌中MOCO生物合成的MOCD或抗生素的治疗进展 目前，由于我们对MoCO生物合成机制的了解有限，因此很难实现这一目标。长期目标 这个项目的目的是提供对病原菌中moco生物合成的机制的理解，以及 在人类身上。目前应用的重点是两种酶(MOAA和MoaC)的作用机制 以鸟嘌呤3‘-三磷酸(GTP)为原料合成MOCO的吡喃喋呤结构。而催化剂 MOAA和MoaC的功能20年来一直不明确，我们最近证明了MOAA 催化GTP生成3‘，8-环二氢-GTP(3’，8-cH2GTP)，而MoaC催化3‘，8-环二氢-GTP 3‘，8-cH_2GTP转化为cPMP。在这一应用中，我们将研究MOAA的催化机理 和MoaC通过以下三个目的在人类和细菌中发挥作用。在目标1中，4Fe-4S的氧化还原功能 MOAA中的集群将在休眠状态和周转期间进行调查，以解决其中一个关键问题 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 并将通过核磁共振、X射线等手段探讨多肽挽救MoCD致突变的机制。 使用细菌和人体酶的结晶学和生化分析。在目标3中，我们将测试共价键 和非共价机理的MoAC催化，并考察了基于机理的抑制的共性 细菌和人类的酶。这项拟议的研究具有重要意义，因为它将为 对MOCO主干形成的见解和未来发展的科学基础 治疗MoCD的生物合成抑制剂和新疗法。

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.