Chemistry and Biology of Bacterial Sulfonucleotide Reductases

摘要 含硫氨基酸L-半胱氨酸对病原菌的毒力和生存是不可或缺的。用于生物合成的硫磺- 论文主要来源于微生物对无机硫酸盐的同化作用。硫酸盐所需的基因- 同质化对病原体的生存是必不可少的，但在人类基因组中不存在，因此代表着潜在的 治疗干预的新靶点。在前一个供资周期中，我们定义了催化周期的主要特征 硫代核苷酸还原酶(SRS)是催化硫酸盐还原的第一步从头开始的酶 半胱氨酸等低硫生物分子的合成。我们的研究提供了基本的见解 硫氧还蛋白(Trxs)-维持蛋白质硫醇还原状态的中央抗氧化酶- 识别它们的细胞目标。我们了解到APS还原酶(APSR)中的铁硫簇在蛋白质合成中起着至关重要的作用 作用于活性中心预组织和底物活化，拓展了对Fe-S PRO催化活性的认识. Teins和缺乏这种辅因子的PAPS还原酶(PAPR)的分化进化。这些洞察力导致了 通过与铁硫金属分配中心的相互作用，以一种新的方式靶向APSR的分子。我们还首先发现了-- 对耐药的临床分离株表现出强大杀菌活性的硫酸盐还原类抑制剂 结核分支杆菌。鉴于sRs在病原菌的氧化应激抗性和毒力中的重要性，我们建议 在这里解决最大的开放问题和最大的未得到满足的需求仍然存在的研究领域：经过充分验证 敏锐地抑制微生物还原硫酸盐同化的基本步骤的化学探针(目标1)；定义 致病Trxs促进硫还原和应对氧化还原应激的非冗余功能(目标2)； 掌握微生物半胱氨酸生物合成中活性硫物种(RSS)的运输和输送方面的知识。 总而言之，此续订应用程序中的实验将提供有关 研究不足的代谢化学-还原硫酸盐同化-在最具破坏性的背景下- 病原体--结核分枝杆菌。我们已经建立了出色的合作关系，并在以下方面有良好的业绩记录 提高生产率，以支持这些努力并确保及时完成。我们的努力将集中于以下领域： 硫代谢在未接种硫化物的病原菌氧化胁迫抗性和毒力中的作用 尽管它们很重要，但得到了充分的关注，最终导致了对 与传染病作斗争。

ABSTRACT The sulfur-containing amino acid, L-cysteine, is indispensable for pathogen virulence and survival. Sulfur for biosyn- thesis is largely derived from the assimilation of inorganic sulfate by microorganisms. Genes required for sulfate as- similation are essential to pathogen survival but absent in the human genome and, therefore, represent potential new targets for therapeutic intervention. In the previous funding cycle, we defined key features in the catalytic cycle of sulfonucleotide reductases (SRs), enzymes that catalyze the first committed step in sulfate reduction for de novo synthesis of cysteine and other reduced-sulfur containing biomolecules. Our studies provided fundamental insights into how thioredoxins (Trxs)–central antioxidant enzymes that maintain protein thiols in their reduced state– recognize their cellular targets. We learned that the iron-sulfur cluster in APS reductase (APSR) plays an essential role active-site preorganization and substrate activation, expanding knowledge on the catalytic activities of Fe-S pro- teins and on the divergent evolution of PAPS reductase (PAPR), which lacks this cofactor. These insights led to molecules that target APSR in a new way, via interaction with the iron-sulfur metallocenter. We also discovered first- in-class inhibitors of sulfate reduction that exhibit potent bactericidal activity against drug-resistant clinical isolates of M. tuberculosis. Given the importance of SRs in pathogen oxidative stress resistance and virulence, we propose here to address research areas where the biggest open questions and greatest unmet needs remain: well-validated chemical probes that acutely inhibitor essential steps in microbial reductive sulfate assimilation (Aim 1); defining the non-redundant functions of pathogenic Trxs to facilitate sulfur reduction and cope with redox stress (Aim 2); and ad- vancing knowledge in trafficking and delivery of reactive sulfur species (RSS) for microbial cysteine biosynthesis. Collectively, the experiments in this renewal application will provide fundamental information on one of the most under-studied metabolic chemistries–reductive sulfate assimilation–in the context of one of the most devastat- ing pathogens–M. tuberculosis. We have established outstanding collaborations, with a proven track record of productivity, in order to support these efforts and ensure timely completion. Our efforts will address areas cen- tral to role of sulfur metabolism in pathogen oxidative stress resistance and virulence that have not received suf- ficient attention despite their importance, ultimately leading to a new level of understanding and leverage in the battle against infectious disease.