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Coenzyme F420-dependent enzymes in mycobacteria

分枝杆菌中辅酶 F420 依赖性酶

基本信息

批准号：
8487357
负责人：
Biswarup Mukhopadhyay
金额：
$ 18.75万
依托单位：
VIRGINIA POLYTECHNIC INST AND ST UNIV
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-06-13 至 2016-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8487357
关键词：
Acids Actinobacteria class Actinomyces Aflatoxins Antibiotics Antineoplastic Agents Archaea Area Bioinformatics Biological Biological Assay Biology Cell Extracts Cell Wall Cell physiology Cholesterol Coenzymes Computer Analysis DNA Photolyase Data Development Disease Electrodes Electrons Environment Enzymes Extreme drug resistant tuberculosis Face Flavins Future Gene Cluster Genomics Genus Mycobacterium Glucosephosphate Dehydrogenase Goals Granuloma Homologous Gene Human Human Resources Immune response Immune system Immunomodulators Inflammatory Investigation Knowledge Leprosy Link Lipids Mediating Metabolism Methanobacteria Multi-Drug Resistance Mycobacterium leprae Mycobacterium tuberculosis Mycolic Acid NR4A1 gene Nocardia Nutrient Open Reading Frames Organism Oxidation-Reduction Oxidoreductase Participant Pathogenesis Physiological Play Preparation Production Proteins Reaction Reducing Agents Research Resistance Role Site Soil Streptomyces Structure System Testing Therapeutic Tuberculosis UV induced DNA damage Waxes Work base carbene cholesterol biosynthesis coenzyme F420 disorder control genetic analysis genome sequencing killings lipid biosynthesis member microorganism mycobacterial nitrosative stress novel pathogen research study resistant strain screening success therapeutic development tuberculosis drugs tuberculosis treatment

项目摘要

DESCRIPTION (provided by applicant): The long-term goal of the proposed project is to define the physiological roles of a subset of the COG2141 enzymes that are dependent on coenzyme F420 and are widespread in the bacterial phylum of Actinobacteria. This phylum includes Mycobacterium tuberculosis and Mycobacterium leprae, which cause tuberculosis (TB) and leprosy, respectively, and Streptomyces, Nocardia, and Actinomyces that produce numerous antibiotics, anticancer agents, and immunomodulators. F420, a deazaflavin coenzyme, was discovered 60 years ago in a mycobacterial species, but its biological roles have been studied mostly in the methanogenic archaea where it was encountered about 30 years ago. This is ironic because the Actinobacteria rather than the methanogens have preponderances of potential F420-utilizing enzymes; even limited work on the mycobacterial F420 metabolism has revealed novel systems. For example, the work by the PI and senior personnel of this proposal show that mycobacteria employ an F420-dependent glucose-6-phosphate dehydrogenase to generate high intracellular levels of reduced F420 (F420H2), which in turn could help to neutralize the nitrosative stress imposed by human immune system. An F420H2-utilizing enzyme helps to unleash the anti-mycobacterial attack from PA-824, a new TB drug. Certain F420-dependent mycobacterial enzymes also degrade aflatoxins. To unravel more novel activities in the putative F420-utilizing enzymes identified by bioinformatic analyses, we are taking an experimental approach. Our focus is on certain mycobacterial COG2141 enzymes that seem to be F420-dependent. Considering that the obligate ground-state hydride transfer function of F420 is similar to that of NAD(P), we are investigating the roles of F420-dependent enzymes in cell wall biosynthesis and cholesterol degradation; these are common sites of action by NAD(P). These selections are also supported by the following deductions. Since the mycobacteria face low redox environments within the human granuloma and soil, they could derive more reducing power from lipid degradation if they use F420 in place of NAD(P); the mid- point redox potential values for F420 and NAD(P) are -360 mV and -320 mV, respectively. Similarly, F420H2 would be a more potent reductant than NAD(P)H for lipid biosynthesis. Our studies have identified three potentially F420-dependent COG2141 enzymes as participants in the synthesis of cell wall lipids (mycolic acids) and waxes (phthiocerol), and degradation of cholesterol in M. tuberculosis; these activities are relevant to TB pathogenesis and can therefore be targeted for developing therapeutics for TB. For detailed structure-function and physiological studies with these enzymes to begin, it is necessary to examine our leads further and strengthen our hypotheses. We therefore propose an R21 project with the following specific aims. Aim 1: To demonstrate that hydroxymycolic acid dehydrogenase and phthiodiolone ketoreductase of M. tuberculosis are F420-dependent enzymes; Aim 2: To examine the possibility of an F420-dependent dehydrogenase assisting cholesterol or lipid degradation in M. tuberculosis.

描述（由申请人提供）：拟议项目的长期目标是确定依赖于辅酶F420并广泛存在于放线菌门的COG 2141酶亚组的生理作用。该门包括分别引起结核病（TB）和麻风病的结核分枝杆菌（Mycobacterium tuberculosis）和麻风分枝杆菌（Mycobacterium leprae），以及产生多种抗生素、抗癌剂和免疫调节剂的链霉菌属（Streptomyces）、诺卡氏菌属（Nocardia）和放线菌属（Actinomyces）。F420是一种脱氮黄素辅酶，60年前在一种分枝杆菌中发现，但其生物学作用主要在产甲烷古菌中研究，大约30年前在那里遇到了它。这是具有讽刺意味的，因为放线菌，而不是产甲烷菌具有优势的潜在的F420利用酶，甚至有限的工作，分枝杆菌F420代谢揭示了新的系统。例如，该提案的主要研究者和高级人员的工作表明，分枝杆菌利用F420依赖性葡萄糖-6-磷酸脱氢酶产生高细胞内水平的还原型F420（F420 H2），这反过来有助于中和人体免疫系统施加的亚硝化应激。一种利用F420 H2的酶有助于释放PA-824（一种新的结核病药物）的抗分枝杆菌攻击。某些F420依赖性分枝杆菌酶也降解黄曲霉毒素。为了揭示更多的新活动，在假定的F420利用酶的生物信息学分析确定，我们正在采取实验方法。我们的重点是某些分枝杆菌COG 2141酶，似乎是F420依赖性。考虑到F420的专性基态氢化物转移函数与NAD（P）的类似，我们正在研究F420依赖性酶在细胞壁生物合成和胆固醇降解中的作用;这些是NAD（P）的常见作用位点。这些选择也得到了以下扣除的支持。由于分枝杆菌在人类肉芽肿和土壤中面临低氧化还原环境，如果它们使用F420代替NAD（P），它们可以从脂质降解中获得更多的还原能力; F420和NAD（P）的中点氧化还原电位值分别为-360 mV和-320 mV。类似地，对于脂质生物合成，F420 H2将是比NAD（P）H更有效的还原剂。我们的研究已经确定了三种潜在的F420依赖性COG 2141酶参与合成细胞壁脂质（分枝菌酸）和蜡（phthiocerol），并降解胆固醇在M。结核病;这些活性与TB发病机理相关，因此可以靶向开发TB的治疗剂。为了开始对这些酶进行详细的结构-功能和生理学研究，有必要进一步检查我们的线索并加强我们的假设。因此，我们提出了一个R21项目，具体目标如下。目的1：研究M.目的2：研究结核分枝杆菌中F420依赖性脱氢酶是否有助于胆固醇或脂质降解。结核