Bacterial lipoate synthesis revisited: novel enzymes, unusual substrates and new evolutionary perspectives

硫辛酸存在于生命的各个领域，参与了中心碳代谢和异化硫氧化的关键反应。在这个八碳饱和脂肪酸中，硫原子取代了酰基链上碳6和碳8的氢原子。在靶蛋白上合成脂酰部分的两种翻译后机制被很好地表征：游离前体的回收或脂肪酸生物合成中间体的重新合成。近年来，这一重要生物分子的组装途径被发现，广泛分布于两个原核结构域。以前，脂酸蛋白连接酶被认为只在脂酸回收中起作用。然而，我们对细菌硫氧化剂的研究表明，它们含有参与蛋白质从头脂酰化的连接酶，从辛酸盐作为自由前体开始。脂酸盐的组装通过两个含SAM结构域的自由基蛋白LipS1和LipS2来完成。对于古生菌柯达卡热球菌，该途径最近已被实验证明，而对细菌领域代表的相应工作仍在等待中。我们的目标是通过解决以下主要问题来详细描述新型细菌脂酸盐组装途径：识别所有参与该途径的酶。2. 性质以及相互作用的新型细菌脂酸组装蛋白，特别是与他们的古细菌对应物的差异。3. 底物范围和在同一生物体中平行运行的脂酰化途径的底物之间的分化机制。4. 新的脂酰化途径的流行和一般重要性，重点是领域细菌。脂酸盐装配机械的起源与发展。几个不同的实验方法将被应用：遗传学研究将包括基因失活和互补的甲变形菌菌丝微生物反硝化。将从反硝化H.参考菌株和突变菌株中纯化和分析天然LbpA2蛋白，使我们能够将单个组装步骤分配给单个酶。对纯重组蛋白进行详细的生化实验将辅以结构分析，从而进一步了解所研究系统的功能。将利用细菌载脂蛋白双酚a蛋白作为天然底物，建立一种结合新酶的脂酸盐体外组装系统。详尽的大规模数据库分析将使用我们新的生物信息学工具HMS-S-S进行。我们打算在完整的生命树上绘制新的脂酸合成途径，从而揭示其分布。最后，将进行系统发育分析，以收集有关新合成途径进化的信息。

Lipoic acid is present in organisms throughout all domains of life and involved in key reactions of central carbon metabolism and dissimilatory sulfur oxidation. In this eight-carbon saturated fatty acid, sulfur atoms replace the hydrogen atoms of carbons 6 and 8 of the acyl chain. Two posttranslational machineries for synthesis of lipoyl moieties on their target proteins are well characterized: Salvage of free precursors or de novo synthesis from intermediates of fatty acid biosynthesis. Recently, a novel pathway for the assembly of this important biomolecule has been discovered, which is widely distributed in both prokaryotic domains. Previously, lipoate:protein ligases had been thought to exclusively operate in lipoate salvage. However, our work on bacterial sulfur oxidizers shows that they contain ligases involved in de novo lipoylation of proteins, starting from octanoate as a free precursor. Lipoate assembly is then completed by the incorporation of sulfur via two radical SAM domain-containing proteins, LipS1 and LipS2. For the archaeon Thermococcus kodakarensis the pathway has recently been experimentally demonstrated, while corresponding work on representatives of the domain Bacteria is still pending. Our goal is to characterize the novel bacterial lipoate assembly pathway in detail by addressing the following major points: 1. Identification of all enzymes involved in the pathway. 2. Properties as well as interactions of the novel bacterial lipoate assembly proteins especially with respect to differences from their archaeal counterparts. 3. Substrate ranges and mechanisms of differentiation between substrates for lipoylation pathways running in parallel in the same organism. 4. Prevalence and general importance of the new lipoylation pathway with a focus on the domain Bacteria. 5. Origin and evolution of lipoate assembly machineries. Several different experimental approaches will be applied: Genetic studies will include gene inactivation and complementation in the Alphaproteobacterium Hyphomicrobium denitrificans. The native LbpA2 protein will be purified and analysed from H. denitrificans reference and mutant strains allowing us to assign individual assembly steps to individual enzymes. Detailed biochemical experiments on the pure recombinant proteins will be complemented by structural analyses and thus provide further insight into the function of the studied system. An in vitro system for lipoate assembly combining the new enzymes will be established using bacterial apo-LbpA proteins as the native substrate. Exhaustive large-scale database analyses will be performed using our new bioinformatic tool, HMS-S-S. We intend to map the novel lipoate synthesis pathway on the complete tree of life thus revealing its distribution. Finally, phylogenetic analyses will be performed in order to gather information on the evolution of the new synthesis pathway.