像牛和羊这样的反刍动物依赖于植物性饲料中碳水化合物的分解,这是由瘤胃中的微生物群落完成的。大约40%的瘤胃微生物群成员属于Prevotellaceae家族,将糖发酵成有机酸,如乙酸盐,丙酸盐,和琥珀酸。这些底物是反刍动物的重要营养素。在牛瘤胃的元蛋白质组分析中,在不同的Prevotella物种中鉴定出与Na易位NADH:醌氧化还原酶(NQR)和醌:富马酸还原酶(QFR)同源的蛋白质。这里,我们表明,通过由NQR和QFR组成的超复合物,厌氧生长的普氏菌中富马酸盐还原为琥珀酸盐与化学渗透能量守恒有关。这种钠易位的NADH:富马酸氧化还原酶(SNFR)超复合物通过蓝色天然PAGE(BN-PAGE)富集,并通过凝胶内酶活性染色和质谱进行表征。高NADH氧化(850nmolmin-1mg-1),醌还原(490nmolmin-1mg-1),和富马酸盐减少(1,200nmolmin-1mg-1)活性,以及高表达水平,证明SNFR代表布氏疟原虫中的电荷分离单元。暴露于不同底物的SNFR的吸收光谱显示,分子内电子从NQR中的黄素腺嘌呤二核苷酸(FAD)辅因子转移到QFR中的血红素b辅因子。SNFR催化NADH和富马酸盐化学计量转化为NAD+和琥珀酸盐。我们认为,在P.bryantii中NAD的再生与电化学梯度的建立密切相关,该梯度通过电子传输磷酸化为ATP合成提供动力。重要性反刍动物的喂养策略旨在优化动物的营养效率,并防止能量损失,如提高甲烷产量。关键是瘤胃微生物群的发酵反应,以普雷沃氏菌属为主。我们表明,通过新描述的由Na转运的NADH:醌氧化还原酶(NQR)和富马酸还原酶(QFR)组成的超复合物,将白蜡菌的琥珀酸形成与NADH氧化和钠梯度形成偶联,代表钠转位NADH:富马酸氧化还原酶(SNFR)超复合物。SNFR是主要的电荷分离模块,产生电化学钠梯度。我们的发现为观察到使用富马酸盐作为饲料添加剂不会显着增加琥珀酸盐的产量提供了线索。或者减少产甲烷,瘤胃中的微生物群落。
Ruminants such as cattle and sheep depend on the breakdown of carbohydrates from plant-based feedstuff, which is accomplished by the microbial community in the rumen. Roughly 40% of the members of the rumen microbiota belong to the family Prevotellaceae, which ferments sugars to organic acids such as acetate, propionate, and succinate. These substrates are important nutrients for the ruminant. In a metaproteome analysis of the rumen of cattle, proteins that are homologous to the Na+-translocating NADH:quinone oxidoreductase (NQR) and the quinone:fumarate reductase (QFR) were identified in different Prevotella species. Here, we show that fumarate reduction to succinate in anaerobically growing Prevotella bryantii is coupled to chemiosmotic energy conservation by a supercomplex composed of NQR and QFR. This sodium-translocating NADH:fumarate oxidoreductase (SNFR) supercomplex was enriched by blue native PAGE (BN-PAGE) and characterized by in-gel enzyme activity staining and mass spectrometry. High NADH oxidation (850 nmol min-1 mg-1), quinone reduction (490 nmol min-1 mg-1), and fumarate reduction (1,200 nmol min-1 mg-1) activities, together with high expression levels, demonstrate that SNFR represents a charge-separating unit in P. bryantii. Absorption spectroscopy of SNFR exposed to different substrates revealed intramolecular electron transfer from the flavin adenine dinucleotide (FAD) cofactor in NQR to heme b cofactors in QFR. SNFR catalyzed the stoichiometric conversion of NADH and fumarate to NAD+ and succinate. We propose that the regeneration of NAD+ in P. bryantii is intimately linked to the buildup of an electrochemical gradient which powers ATP synthesis by electron transport phosphorylation. IMPORTANCE Feeding strategies for ruminants are designed to optimize nutrient efficiency for animals and to prevent energy losses like enhanced methane production. Key to this are the fermentative reactions of the rumen microbiota, dominated by Prevotella spp. We show that succinate formation by P. bryantii is coupled to NADH oxidation and sodium gradient formation by a newly described supercomplex consisting of Na+-translocating NADH:quinone oxidoreductase (NQR) and fumarate reductase (QFR), representing the sodium-translocating NADH:fumarate oxidoreductase (SNFR) supercomplex. SNFR is the major charge-separating module, generating an electrochemical sodium gradient in P. bryantii. Our findings offer clues to the observation that use of fumarate as feed additive does not significantly increase succinate production, or decrease methanogenesis, by the microbial community in the rumen.