Novel lipoate-binding proteins and their role in sulfur oxidation

硫辛酸（1,2-二硫烷-3-戊酸）是一种高度保守的有机硫辅因子，存在于几乎所有的原核生物和真核生物中。辅助因子对参与氧化和单碳代谢的几个关键酶的功能至关重要。到目前为止，仅鉴定了五种脂酸依赖的多酶复合物：三种α-酮酸脱氢酶（如丙酮酸脱氢酶），乙酰脱氢酶和甘氨酸裂解复合物。虽然硫辛酸的存在已经被发现了60多年，但我们最近的工作揭示了这种辅助因子的一种意想不到的代谢功能，这种功能与它在中枢代谢中的典型作用明显不同。我们证明了一种新的脂酸结合蛋白（LbpA）在异化硫氧化的新途径中起着不可或缺的作用。杂二硫还原酶（Hdr）样复合体是这一途径的核心参与者。它发生在一个巨大的生物群体中，包括与生物技术和环境相关的细菌，如挥发性有机硫化合物降解的甲变形杆菌菌丝微生物反硝化菌和许多化能自养细菌和古细菌。在目前已知的所有情况下，脂酸盐作为一个摆动臂，在不同亚基的活性位点之间引导结合的底物。在催化过程中，氧化脂酰胺与还原二氢脂酰胺之间的脂酸分子内二硫键循环，二氢脂酰胺再氧化释放的电子可通过二氢脂酰胺脱氢酶直接转移到NAD+上。如果脂酸结合蛋白在依赖于NADH的硫氧化中发挥类似的功能，那么至少有一些在这里释放的电子可以直接用于NADH的形成。这样的反应将大大减少硫氧化型自养生物对能量要求高的反向电子流的需求。对于这些令人兴奋的建议，目前还没有直接的生物化学或遗传学证据。在本项目中，我们旨在填补这一空白，并打算澄清新型脂酸结合蛋白在硫氧化过程中的确切作用。以下主要问题将被解决：[1]LbpA蛋白是否作为硫结合实体将底物呈现给新的异二硫还原酶样硫氧化络合物的不同催化位点？在类hdr络合物的催化循环中，脂酸盐是否在其氧化和还原形式之间切换，使得在hdr驱动的硫氧化过程中释放的至少部分电子可以直接用于生成NADH？还有哪些硫转移酶参与了这个过程？

Lipoic acid (1,2-dithiolane-3-pentanoic acid) is a highly conserved organosulfur cofactor found in almost all prokaryotic and eukaroytic organisms. The cofactor is essential for the function of several key enzymes involved in oxidative and one-carbon metabolism. So far, only five lipoate-dependent multi¬enzyme complexes have been characterized: three α-ketoacid dehydrogenases (e.g. pyruvate dehydrogenase), acetoin dehydrogenase and the glycine cleavage complex.Although the existence of lipoic acid has been known for more than sixty years, our recent work revealed an unexpected metabolic function for this cofactor that is markedly different from its canonical roles in central metabolism. We proved that a novel lipoate-binding proteins (LbpA) act as indispensable components of a new pathway of dissimilatory sulfur oxidation. A heterodisulfide reductase (Hdr)-like complex is a central player in this pathway. It occurs in a huge organism group that includes biotechnologically and environmentally relevant bacteria like the volatile organic sulfur compound degrading Alphaproteobacerium Hyphomicrobium denitrificans and many chemolithoautotrophic bacteria and archaea. In all cases known so far, lipoate acts as a swinging arm that channels bound substrate between the active sites of different subunits. During catalysis, the intramolecular disulfide bond of lipoate cycles between oxidized lipoamide and reduced dihydrolipoamide and the electrons released upon reoxidation of dihydroliponamide can be directly transferred onto NAD+ via dihydroliponamide dehydrogenase. If lipoate-binding proteins perform similar functions in Hdr-dependent sulfur oxidation, then at least some of the electrons released here could be used directly for the formation of NADH. Such a reaction would considerably reduce the need for energy-demanding reverse electron flow in sulfur-oxidizing lithoautotrophs. Direct biochemical or genetic evidence for these exciting suggestions is currently not available. In this project, we aim at at filling this gap and intend to clarify the exact role of the novel lipoate binding proteins during sulfur oxidation. The following major questions will be addressed: [1] Do LbpA proteins function as sulfur-binding entities presenting substrate to different catalytic sites of the new heterodisulfide reductase-like sulfur-oxidizing complex? [2] Does lipoate switch between its oxidized and reduced forms during the catalytic cycle of the Hdr-like complex such that at least part of the electrons released during Hdr-driven sulfur oxidation can be directly used for generation of NADH? [3] Which other sulfur transferases are involved in the process?