Functional characterization of the formate dehydrogenase orthologue and hydrogenases in organohalide respiration by Dehalococcoides mccartyi

脱卤球菌属物种进行有机卤化物呼吸的一个主要电子供体是氢；然而，一般对有机卤化物呼吸的其他潜在电子供体知之甚少。脱卤球菌属CBDB1菌株不能使用甲酸作为电子供体，但它能合成甲酸脱氢酶(FDH)作为其最丰富的蛋白质之一。这一事实和其他事实一起表明，这种酶具有不同的功能。因此，这项提议的目的之一是确定这种类FDH酶在有机卤化物呼吸中的生理功能。我们将使用生化、生理和分子生物学相结合的方法，结合在大肠杆菌中的异源表达，来鉴定类FDH蛋白在嗜盐球藻中的作用。脱氢球菌CBDB1株的基因组也有能力合成大约5种不同类型的多亚单位氢酶；其中4种被预测为[NiFe]-氢酶。第五个被预测为[FeFe]-氢酶(Hym)，尽管它与复杂的I组分也有很强的序列相似性。我们的目标是确定菌株CBDB1通过Hym是否像消耗氢一样释放和消耗氢，并确定单个氢酶对有机卤化物呼吸过程中氢分压和电子受体光谱的响应对生长的贡献。

A major electron donor for organohalide respiration by Dehalococcoides species is hydrogen; however, little is generally known concerning other potential electron donors for organohalide respiration. Dehalococcoides strain CBDB1 cannot use formate as an electron donor yet it synthesizes a putative formate dehydrogenase (Fdh) as one of its most abundant proteins. This fact, along with others, indicates that the enzyme has a different function. One of the aims of this proposal is therefore, to determine the physiological function of this Fdh-like enzyme in organohalide respiration. We will use combined biochemical, physiological and molecular biological approaches, together with heterologous expression in E. coli, to identify the role of the Fdh-like protein in Dehalococcoides spp. The genome of Dehalococcoides strain CBDB1 also has the capacity to synthesise probably up to 5 different types of multisubunit hydrogenase; 4 of these are predicted to be [NiFe]-hydrogenases. The fifth is predicted to be a [FeFe]-hydrogenase (Hym) although it also has strong sequence similarity to complex I components. Our aim is to resolve whether strain CBDB1 via Hym evolves as well as consumes hydrogen and to determine the contribution of the individual hydrogenases to growth in response to hydrogen partial pressure and electron acceptor spectrum during organohalide respiration.