Regulation of substrate dependent expression and activity from dehalogenating enzymes in Dehalococcoides mccartyi strain CBDB1

有机卤化物呼吸的生物化学仍然难以捉摸，因为一些能够进行有机卤化物呼吸的物种不能提供足够的生物量来进行纯化酶的经典生物化学。多肽光谱灵敏度的提高和现有的基因组信息将加强对有机卤化物呼吸器生化原理的研究。多食硫磺螺杆菌(Sulfurospirillum Multivorans)，脱卤球菌属(Dehalodiccoides sp.)CBDB1、DCMB5和BTF08将由次级项目伙伴1、2、3和4在本研究股的框架内提供，并用不同的电子供体、电子受体和碳源培养。该种的有限生物量。需要对蛋白质提取方案和高分辨率质谱仪的测量进行优化。鸟枪式蛋白质组学分析将提供对所涉及的蛋白质的更深入的了解，从而提供相应的生化途径。在不同的有机卤化物呼吸物种中，发现了种类繁多的还原脱卤酶基因，但既不了解它们的底物特异性，也不了解它们的合成对底物的响应规律。选择反应监测-质谱仪(SRM-MS)将对还原脱卤酶的不同基因产物进行准确的检测和定量。这种有针对性的蛋白质组学方法使用重同位素标记的标准多肽，通过QTRAP-MS对消化蛋白质提取物中感兴趣的多肽进行定量。这种特定的定量将使人们深入了解底物、培养条件和所涉及的生化途径之间的关系，以及同源还原脱卤酶的表达。

The biochemistry of organohalide respiration is still elusive because some of the species capable of it do not provide sufficient biomass for classical biochemistry on purified enzymes. The improvement of sensitivity in mass spectrometry of peptides together with available genomic information will enhance the study of the biochemical principles of organohalide respirers. The species Sulfurospirillum multivorans, Dehalococcoides sp. CBDB1, DCMB5 and BTF08 will be provided by the subproject partners 1, 2, 3 and 4 within the framework of this Research Unit and cultivated with different electron donors, electron acceptors and carbon sources. The limited biomass of the Dehalococcoides spp. requires optimisation of both protein extraction protocols and measurement by high-resolution mass spectrometers. Shotgun proteomic analyses will provide a deeper insight into the proteins involved and thus the corresponding biochemical pathways. Among the different organohalide respiring species a wide variety of reductive dehalogenase genes are found and neither their substrate specificity nor the regulation of their synthesis in response to the substrate is understood. An accurate detection and quantification of the different gene products of the reductive dehalogenases will be performed by selected reaction monitoring – mass spectrometry (srm-MS). This targeted proteomics approach uses standard peptides labelled with heavy isotopes for quantification of peptides of interest in digested protein extracts by qtrap-MS. The specific quantification will yield insights into the correlation between substrate, culture conditions and the biochemical pathways involved as well as the expression of orthologous reductive dehalogenases.