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

硫辛酸存在于生物体的所有生命领域，并参与中心碳代谢和异化硫氧化的关键反应。在这种八碳饱和脂肪酸中，硫原子取代酰基链的碳6和8的氢原子。两个翻译后机器合成的硫辛酰部分的目标蛋白质的特点是：救助的游离前体或从头合成脂肪酸生物合成的中间体。最近，发现了一种新的组装这一重要生物分子的途径，它广泛分布于原核生物的两个结构域。以前，人们认为硫辛酸：蛋白质连接酶只在硫辛酸补救中起作用。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，从辛酸作为游离前体开始。Lipoate组装然后通过经由两个含自由基SAM结构域的蛋白质LipS 1和LipS 2掺入硫来完成。对于古菌Thermococcus kodakarensis的途径最近已被实验证明，而相应的工作的代表域细菌仍悬而未决。我们的目标是详细描述新的细菌硫辛酸组装途径，解决以下几个主要问题：1。鉴定参与该途径的所有酶。2.特性以及新的细菌硫辛酸组装蛋白的相互作用，特别是相对于他们的古细菌对应的差异。3.底物范围和在同一生物体中平行运行的脂酰化途径底物之间的差异机制。4.新的脂酰化途径的普遍性和普遍重要性，重点是细菌领域。5.脂肪酸酯装配机械的起源与发展。将应用几种不同的实验方法：遗传研究将包括在Alphaproteobacterium Hyphomicrobium acidificans中的基因失活和互补。天然LbpA 2蛋白将从H.参考菌株和突变菌株使我们能够将单个组装步骤分配给单个酶。对纯重组蛋白的详细生化实验将通过结构分析来补充，从而进一步了解所研究系统的功能。将建立一个在体外系统的新酶的组合，使用细菌载脂蛋白LbpA蛋白作为天然底物的硫辛酸组装。将使用我们的新生物信息学工具HMS-S-S进行详尽的大规模数据库分析。我们打算在完整的生命树上绘制新的硫辛酸合成途径，从而揭示其分布。最后，将进行系统发育分析，以收集新的合成途径的演变信息。

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.