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

Dehalococcoides物种呼吸有机卤化物的主要电子供体是氢；然而，对于有机卤化物呼吸作用的其他潜在电子供体，一般知之甚少。dehalococoides菌株CBDB1不能以甲酸作为电子供体，但它合成了一种假定的甲酸脱氢酶（Fdh），作为其最丰富的蛋白质之一。这一事实以及其他事实表明，这种酶具有不同的功能。因此，本提案的目的之一是确定这种fdh样酶在有机卤化物呼吸中的生理功能。我们将采用生化、生理和分子生物学相结合的方法，结合大肠杆菌中的异源表达，来确定fdh样蛋白在Dehalococcoides spp中的作用。Dehalococcoides菌株CBDB1的基因组也具有合成可能多达5种不同类型的多亚基氢化酶的能力；其中4个被预测为[NiFe]氢化酶。第五个预测是[FeFe]-氢化酶（Hym），尽管它也与复合物I组分具有很强的序列相似性。我们的目的是确定菌株CBDB1是否通过Hym进化和消耗氢，并确定个体氢化酶对生长的贡献，以响应氢分压和有机卤化物呼吸过程中的电子受体谱。

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.