Coenzyme F420-dependent enzymes in mycobacteria

分枝杆菌中辅酶 F420 依赖性酶

• 批准号: 8386085
• 负责人: Biswarup Mukhopadhyay
• 金额: $ 23.61万
• 依托单位: VIRGINIA POLYTECHNIC INST AND ST UNIV
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-13 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8386085
• 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; Screening procedure; 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; success; therapeutic development; tuberculosis drugs; tuberculosis treatment

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. PUBLIC HEALTH RELEVANCE: The proposed investigation concerns cellular functions of a relatively unstudied group of enzymes that utilizes a deazaflavin coenzyme and potentially plays key roles in the development of tuberculosis (TB) and the production of antibiotics and other bioactive compounds by certain soil microorganisms. The current knowledge about these proteins comes mostly from computer analysis of genome sequences and theoretical deductions. The proposed work will follow newly obtained leads and test some of the above-mentioned theoretical predictions experimentally, thereby enabling future detailed studies on the development of therapeutics for TB and other diseases.

描述（由申请人提供）：拟议项目的长期目标是确定COG2141酶的一个亚群的生理作用，该亚群依赖于辅酶F420，在放线菌门细菌中广泛存在。这一门包括结核分枝杆菌和麻风分枝杆菌，它们分别引起结核病和麻风，以及链霉菌、诺卡菌和放线菌，它们产生大量抗生素、抗癌药物和免疫调节剂。F420是一种去氮黄素辅酶，60年前在一种分枝杆菌中被发现，但其生物学作用主要是在产甲烷的古细菌中研究的，大约30年前在那里发现了它。这是具有讽刺意味的，因为放线菌而不是产甲烷菌具有潜在的f420利用酶的优势；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)，这反过来可以帮助中和人体免疫系统施加的亚硝化应激。一种利用f420h2的酶有助于释放抗结核药物PA-824的分枝杆菌攻击。某些依赖f420的分枝杆菌酶也能降解黄曲霉毒素。为了利用生物信息学分析确定的酶来揭示f420中更多的新活性，我们正在采取实验方法。我们的重点是某些分枝杆菌COG2141酶，这些酶似乎依赖于f420。考虑到F420的专性基态氢化物传递功能与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)的共同作用位点。这些选择也得到下列推论的支持。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。同样，F420H2在脂质生物合成中是比NAD(P)H更有效的还原剂。我们的研究已经确定了三种可能依赖于f420的COG2141酶，它们参与了结核分枝杆菌细胞壁脂质（霉菌酸）和蜡质（硫代酚）的合成以及胆固醇的降解；这些活性与结核病发病机制有关，因此可以作为开发结核病治疗方法的目标。为了开始对这些酶进行详细的结构功能和生理研究，有必要进一步检查我们的线索并加强我们的假设。因此，我们提出一个R21项目，具体目标如下：目的1：证实结核分枝杆菌的羟基霉菌酸脱氢酶和噻唑二酮酮还原酶是f420依赖性酶；目的2：研究f420依赖性脱氢酶在结核分枝杆菌中协助胆固醇或脂质降解的可能性。