Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond, VA, USA.
Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, IL, USA; Microbiome Metabolic Engineering Theme, Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA.
Biochim Biophys Acta Mol Cell Biol Lipids. 2018 Mar;1863(3):276-283. doi: 10.1016/j.bbalip.2017.12.001. Epub 2017 Dec 5.
The multi-step bile acid 7α-dehydroxylating pathway by which a few species of Clostridium convert host primary bile acids to toxic secondary bile acids is of great importance to gut microbiome structure and host physiology and disease. While genes in the oxidative arm of the 7α-dehydroxylating pathway have been identified, genes in the reductive arm of the pathway are still obscure.
We identified a candidate flavoprotein-encoding gene predicted to metabolize steroids. This gene was cloned and overexpressed in E. coli and affinity purified. Reaction substrate and product were separated by thin layer chromatography and identified by liquid chromatograph mass spectrometry-ion trap-time of flight (LCMS-IT-TOF). Phylogenetic analysis of the amino acid sequence was performed.
We report the identification of a gene encoding a flavoprotein (EDS08212.1) involved in secondary bile acid metabolism by Clostridium scindens ATCC 35704 and related species. Purified rEDS08212.1 catalyzed formation of a product from 3-dehydro-deoxycholic acid that UPLC-IT-TOF-MS analysis suggests loses 4amu. Our phylogeny identified this gene in other bile acid 7α-dehydroxylating bacteria.
These data suggest formation of a product, 3-dehydro-4,6-deoxycholic acid, a recognized intermediate in the reductive arm of bile acid 7α-dehydroxylation pathway and the first report of a gene in the reductive arm of the bile acid 7α-dehydroxylating pathway.
少数梭菌通过多步胆汁酸 7α-脱羟途径将宿主初级胆汁酸转化为有毒的次级胆汁酸，这对肠道微生物组结构和宿主生理及疾病非常重要。虽然已经鉴定出 7α-脱羟途径氧化臂中的基因，但该途径的还原臂中的基因仍然不清楚。
我们鉴定出一个编码预测代谢甾体的黄素蛋白的候选基因。该基因在大肠杆菌中克隆并过表达，并进行亲和纯化。通过薄层层析分离反应底物和产物，并通过液相色谱-质谱-离子阱-飞行时间（LCMS-IT-TOF）进行鉴定。对氨基酸序列进行系统发育分析。
我们报告了鉴定编码参与次级胆汁酸代谢的黄素蛋白（EDS08212.1）的基因，该基因由 Clostridium scindens ATCC 35704 和相关物种编码。纯化的 rEDS08212.1 催化 3-脱氢去氧胆酸形成产物，UPLC-IT-TOF-MS 分析表明该产物失去 4amu。我们的系统发育分析在其他胆汁酸 7α-脱羟细菌中鉴定出该基因。
这些数据表明形成了一种产物，3-脱氢-4,6-去氧胆酸，这是胆汁酸 7α-脱羟途径还原臂中的一种公认中间体，也是胆汁酸 7α-脱羟途径还原臂中基因的首次报道。
