Coenzyme F420-dependent enzymes in mycobacteria

描述(由申请人提供)：拟议项目的长期目标是确定COG2141酶的子集的生理作用，这些酶依赖于辅酶F420，广泛存在于放线杆菌的细菌门中。这一门包括分别导致结核病和麻风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、诺卡氏菌和放线菌。F420是一种去氮黄素辅酶，60年前在一个分枝杆菌物种中被发现，但它的生物学作用主要是在大约30年前遇到它的产甲烷古菌中进行的研究。这是具有讽刺意味的，因为放线杆菌而不是产甲烷菌具有潜在的利用F420的酶的优势；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，PI和这项提案的高级人员的工作表明，分枝杆菌利用依赖于F420的葡萄糖-6-磷酸脱氢酶来产生高水平的还原F420(F420H2)，这反过来又有助于中和人类免疫系统施加的亚硝化应激。一种利用F420H2的酶有助于释放结核病新药PA-824的抗分枝杆菌攻击。某些依赖F420的分枝杆菌酶也能降解黄曲霉毒素。为了揭开通过生物信息学分析确定的假定的F420利用酶的更多新的活性，我们正在采取实验方法。我们的重点是某些分枝杆菌COG2141酶，这些酶似乎是F420依赖的。考虑到F420的专有基态氢化物转移功能与NAD(P)相似，我们正在研究F420依赖的酶在细胞壁生物合成和胆固醇降解中的作用；这些都是NAD(P)的共同作用部位。这些选择还受到以下扣减的支持。由于分枝杆菌在人肉芽肿和土壤中面临低氧化还原环境，如果用F420取代NAD(P)，它们可以从脂肪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。类似地，F420H2是一种比NAD(P)H更有效的脂肪生物合成还原剂。我们的研究已经确定了三种可能依赖于F420的COG2141酶参与了结核分枝杆菌细胞壁脂(真菌酸)和蜡质(邻苯三酚)的合成以及胆固醇的降解；这些活性与结核病的发病机制有关，因此可以作为开发结核病治疗药物的靶点。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，我们提出了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霉菌酸脱氢酶和硫代二醇酮还原酶是依赖于F420的酶；目的2：研究依赖于F420的脱氢酶在结核分枝杆菌中协助胆固醇或脂肪降解的可能性。

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.