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定义依赖于辅酶F420的COG2141酶子集的生理作用，并在静脉细菌的细菌性门内广泛存在。该门包括结核分枝杆菌和麻风分枝杆菌，分别引起结核病（TB）和麻风病，以及链霉菌，Nocardia和放线菌，产生许多抗生素，抗癌药物，抗癌药物和免疫调节剂。 F420是Deazaflavin辅酶，在60年前在分枝杆菌中发现，但其生物学作用主要是在大约30年前遇到的甲烷古细菌中研究的。这是具有讽刺意味的，因为放线菌而不是甲烷剂具有潜在的F420氧化酶的优势。即使在分枝杆菌F420代谢上的工作有限，也揭示了新型系统。例如，PI和该提案的高级人员的工作表明，分枝杆菌采用F420依赖性的6-磷酸葡萄糖-6-磷酸脱氢酶产生高细胞内降低的F420（F420H2），这反过来又可以帮助中和人类免疫系统中和硝基化压力。 F420H2利用酶有助于从新的结核病药物PA-824中释放抗细菌攻击。某些依赖F420的分枝杆菌也降解了黄曲霉毒素。为了揭示通过生物信息学分析确定的假定F420利用酶的新活性，我们正在采用一种实验方法。我们的重点是某些似乎依赖F420的分枝杆菌COG2141酶。考虑到F420的强制地基氢化物转移功能与NAD（P）相似，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）的共同作用部位。这些选择也得到以下扣除的支持。由于分枝杆菌在人肉芽肿和土壤中面临较低的氧化还原环境，因此，如果使用F420代替NAD（P），它们可能会从脂质降解中得出更多的功率。 F420和NAD（P）的中点氧化还原电势值分别为-360 mV和-320 mV。同样，对于脂质生物合成，F420H2将比NAD（P）H更有效。我们的研究已经确定了三种潜在的F420依赖性COG2141酶是细胞壁脂质（霉菌酸）和蜡（磷酸酚）合成的参与者，以及结核分枝杆菌中胆固醇的降解。这些活性与结核病发病机理有关，因此可以针对开发结核病的治疗剂。为了开始使用这些酶开始的详细结构功能和生理研究，有必要进一步检查我们的潜在客户并加强我们的假设。因此，我们提出一个具有以下特定目标的R21项目。目的1：证明结核分枝杆菌的羟基甲酸脱氢酶和邻苯二酚酮酮是F420依赖性酶；目的2：检查辅助胆固醇或脂质降解的F420依赖性脱氢酶的可能性。