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 COG2141 酶子集的生理作用，这些酶依赖于辅酶 F420，并且广泛存在于放线菌细菌门中。该门包括分别引起结核病 (TB) 和麻风病的结核分枝杆菌和麻风分枝杆菌，以及产生大量抗生素、抗癌剂和免疫调节剂的链霉菌属、诺卡氏菌属和放线菌属。 F420 是一种脱氮黄素辅酶，60 年前在一种分枝杆菌中被发现，但其生物学作用主要在大约 30 年前发现的产甲烷古菌中进行研究。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 F420 利用酶；即使对分枝杆菌 F420 代谢的有限研究也揭示了新的系统。例如，该提案的 PI 和高级人员的工作表明，分枝杆菌利用 F420 依赖性葡萄糖-6-磷酸脱氢酶在细胞内产生高水平的还原 F420 (F420H2)，这反过来又有助于中和亚硝化应激由人体免疫系统强加的。 F420H2 利用酶有助于释放新型结核病药物 PA-824 的抗分枝杆菌攻击。某些 F420 依赖性分枝杆菌酶也能降解黄曲霉毒素。为了揭示通过生物信息学分析确定的假定的 F420 利用酶的更多新活性，我们正在采取实验方法。我们的重点是某些似乎依赖于 F420 的分枝杆菌 COG2141 酶。考虑到F420的专性基态氢化物转移功能与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) 的常见作用位点。这些选择也得到以下推论的支持。由于分枝杆菌面临人类肉芽肿和土壤内的低氧化还原环境，如果使用 F420 代替 NAD(P)，它们可以从脂质降解中获得更大的还原能力； F420 和 NAD(P) 的中点氧化还原电位值分别为 -360 mV 和 -320 mV。同样，对于脂质生物合成，F420H2 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 COG2141 酶参与结核分枝杆菌细胞壁脂质（分枝菌酸）和蜡（phthiocerol）的合成以及胆固醇的降解；这些活动与结核病发病机制相关，因此可以作为开发结核病疗法的目标。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，我们提出了一个具有以下具体目标的 R21 项目。目的1：证明结核分枝杆菌的羟基分枝菌酸脱氢酶和邻苯二酚酮还原酶是F420依赖性酶；目标 2：检查 F420 依赖性脱氢酶协助结核分枝杆菌中胆固醇或脂质降解的可能性。