Understanding the diverse biochemistry of the chlorite dismutase family: from O2 to heme

 说明(申请人提供)：血红素是有氧生命和细胞呼吸所必需的。真核细胞制造血红素的途径已经知道一段时间了。相比之下，原核生物的血红素生物合成则更难描述。最近，对于革兰氏阳性细菌，一种填补所有剩余空白的血红素生物合成途径被提出。这是一组有机体，包括许多威胁公共卫生和生物防御的重要病原体，如MRSA、结核病、炭疽和鼠疫的病原体。这条路径与经典路径的不同之处在于它的最后三步，最大的起点是它的终点处。最后一步是由一种名为hemQs的酶催化的双氧化脱羧反应：一种新的亚型亚型的亚氯酸盐歧化酶(Cld)。后者是血红素酶，可以使高氯酸盐呼吸的亚氯酸盐最终产物解毒，将其转化为氯离子到氧气。这项研究的初始阶段对高氯酸盐呼吸器和非呼吸性病原体产生O2的ClD的结构、机制和生物学进行了严格的描述。利用通过在ClD上的工作而收集的工具、见解和科学团队，本提案旨在提供个人级别的hemQ功能的描述 分子，并延伸到细胞环境。作为初步工作，已经产生了一株金黄色葡萄球菌hemQ株，并被证明是一种血红素营养缺陷型和小菌落变异体(SCV)：一种与细胞内持久性和抗生素耐药性相关的表型。同时，金黄色葡萄球菌的hemQ酶被证明可以氧化脱羧基辅酶III的四个丙酸侧链中的两个，这一反应严格依赖于过氧化氢。重点放在金黄色葡萄球菌系统上，目标1是了解hemQ如何结合和激活辅基蛋白进行氧化脱羧化，产生SahemQ与其底物(辅基血红素III)、中间体(Harderoheme)和产物(Heme B)的复合体的结构和能量模型。第二个目的是测试一个反应的机理，在这个反应中，辅基素既是过氧化的底物，又是辅助因子。时间分辨和动力学同位素方法将被用来检验一系列关于铁-氢过氧基中间体和分子内氢原子转移的假设。最后，Aim 3使用遗传学、基于细胞的和生物化学方法来理解hemQ在细胞和进化背景下的功能。我们预计拟议工作的完成将确定一条途径的最终步骤，这条途径对有氧生命绝对基础，对强健的病原体生长至关重要，并在临床上与持久性和抗生素耐药性的发展有关。

 DESCRIPTION (provided by applicant): Heme is essential for aerobic life and cellular respiration. The pathway by which eukaryotic cells make heme has been known for some time. Prokaryotic heme biosynthesis, by contrast, has been harder to describe. Recently, a pathway for heme biosynthesis that fills all the remaining gaps has been proposed for Gram- positive bacteria. This is a group of organisms that includes numerous important pathogens that are threats to public health and biodefense, such as the causative agents of MRSA, TB, anthrax, and plague. The pathway differs from the canonical one in its final three steps, with the greatest departure at its terminus. The last step is a double oxidative decarboxylation catalyzed by enzymes known as HemQs: a novel subtype of chlorite dismutases (Clds). The latter are heme enzymes that detoxify the chlorite end product of perchlorate respiration, converting it to Cl- to O2. The initial phase of this research resulted in a rigorous description of the structure, mechanism, and biology of O2-generating Clds from both perchlorate respirers and non-respiring pathogens. Leveraging the tools, insights, and scientific team assembled via work on Clds, this proposal aims at providing a description of HemQ function at the level of the individual molecule and extending to the cellular context. As preliminary work, a hemQ strain of Staphylococcus aureus has been generated and shown to be a heme auxotroph and small colony variant (SCV): a phenotype associated with intracellular persistence and antibiotic resistance. In tandem, the HemQ enzyme from S. aureus has been shown to oxidatively decarboxylate two of the four propionate side chains of coproheme III, in a reaction that depends strictly H2O2. Focusing on the S. aureus system, Aim 1 is to understand how HemQ binds and activates coproheme toward oxidative decarboxylation, producing structural and energetic models of SaHemQ in complex with its substrate (coproheme III), intermediate (harderoheme) and product (heme b). Aim 2 is to test a mechanism for HemQ's reaction, in which coproheme is both substrate and cofactor in the peroxidation. Time-resolved and kinetic isotope methods will be used to examine a series of hypotheses invoking a ferric-hydroperoxy intermediate and intramolecular hydrogen atom transfer. Finally, aim 3 uses genetic, cell-based, and biochemical methods to understand HemQ's function in the context of the cell and evolution. We expect completion of the proposed work to define the ultimate step of a pathway that is absolutely fundamental to aerobic life, essential for robust pathogenic growth, and clinically connected to the development of persistence and antibiotic resistance.