甘氨酸自由基酶IseG在肠道菌群C、N、S和能量代谢中作用的研究

Sulfate and sulfite reducing bacteria (SSRB) use sulfite as the electron acceptor for respiration, producing toxic H2S linked to colon cancer. Taurine is the main source of sulfite in human gut. However, how SSRB obtain sulfite from taurine is a mystery. Our research group recently discovered a novel glycyl radical enzyme that catalyzes the C-S bond cleavage of isethionate. We named this enzyme IseG. Furthermore, the entire metabolic pathway was unraveled: A transaminase converts taurine into sulfoacetaldehyde, which is then reduced by a novel enzyme (named by us as TauF) generating isethionate. We thus solved the crystal structure of IseG with isethionate bound to its active site. With structural information, we were able to identify the critical conserved residues in the active site and find that IseG is prevalent in bacteria, especially SSRB. We propose that bacteria use IseG and TauF for various purposes including carbon, nitrogen, sulfur acquisition and energy production. Based on our previous biochemical and bioinformatics finding, a cross feeding model was proposed: Bifidobacteria obtain nitrogen from taurine and secret isethionate, which is then uptaken and cleaved by SSRB producing sulfite, as their respiration electron acceptor. To demonstrate this, we will clone, express and purify recombinant proteins including IseG, TauF and respective transporters with different origins and characterize them in vitro. In addition, we will use defined media with either taurine or isethionate to cultivate wild type and mutant SSRB, Clostridium and Bifidobacterium. To prove this taurine partaking model, we will perform sequential culturing experiments as well as coinoculation of both bacteria in germ free mice followed by RT-PCR of the stool samples to study the growth of these bacteria in vivo.

硫还原菌无氧呼吸产生硫化氢，是导致结肠癌的因素。其呼吸作用电子受体亚硫酸在肠道中主要来源于牛磺酸，但机理未知。课题组前期发现新的代谢通路：牛磺酸经转氨酶作用产生磺酸乙醛，后经课题组发现并命名的TauF还原成羟乙磺酸，再经课题组发现并命名的首个催化C-S键断裂的甘氨酸自由基酶IseG催化生成乙醛和亚硫酸。课题组解析了IseG底物复合物结构，根据IseG活性中心的关键氨基酸残基和生物信息学研究，发现其在很多细菌中存在。课题组提出了肠道中不同细菌互惠：双歧杆菌摄取牛磺酸的氮，提供羟乙磺酸给含IseG的硫还原菌作为呼吸作用电子受体。课题组拟表达纯化不同物种的IseG和TauF，研究其催化功能和机理；用牛磺酸或羟乙磺酸作为唯一的碳、氮、硫源体外培养肠道菌，研究这类酶的生理功能，并通过体外共培养以及共接种无菌老鼠结合遗传操作的方式来研究这些菌的生态关系，为针对肠道菌群的药物开发提供精准靶点。

课题组严格按照项目申请书的实验设计方案，对甘氨酸自由基酶IseG在肠道菌群C、N、S和能量代谢中的作用开展了全面的研究，发现了一条新的牛磺酸代谢途径：牛磺酸经转氨酶Tpa作用产生磺酸乙醛，后经课题组发现并命名的脱氢酶TauF还原成羟乙磺酸，再经课题组发现并命名的首个催化C-S键断裂的甘氨酸自由基酶IseG催化生成乙醛和亚硫酸。相关工作已经发表在Nature Communications, Biochemical Journal, Applied & Environmental Microbiology, Bioscience Reports等主流学术期刊上。除此之外，我们还发现了多条微生物初级代谢途径，1）阐明了厌氧菌牛磺酸、络氨酸、磺基葡萄糖的降解途径和自由基酶催化机理，为癌症治疗和新药研发提供新靶标；2）发现了微生物嘧啶和嘌呤降解的新通路，开发了基于益生菌的痛风治疗方案；3）解析了噬菌体二氨基嘌呤（Z）完全取代腺嘌呤基因组的合成机制，拓展了Watson-Crick碱基配对原则和生物遗传信息传递中心法则。发现酶促新反应，了解厌氧生物生存的机制，为开发专门针对厌氧细菌的新型抗生素提供靶点和理论实验基础。相关的工作分别发表在Science, Proc Natl Acad Sci, J. Am. Chem. Soc., and ACS catalysis等主流学术期刊上。在本基金的支持下总共发表SCI论文21篇，获得授权专利1项，已申请并进入实审专利4项。培养了一名博士后、三名博士生和十名硕士研究生。工作成果受到全球媒体的广泛关注和专题报道。具有应用价值的知识产权目前正在与企业洽谈授权、转让。