Corrinoid cofactor biosynthesis and insertion into reductive dehalogenases of anaerobic bacteria

类咕啉辅因子在蛋白质辅助的多步生物合成途径中形成。一种独特的类咕啉，norpseudo-B12，是革兰氏阴性厌氧菌多食硫杆菌四氯乙烯还原脱卤酶（PceA）的辅因子。从革兰氏阳性厌氧菌Desulfitobacterium Rugniense菌株PCE-S纯化的PceA含有一组不同的类咕啉辅因子。该项目的主要目标是确定和表征不同的corrinoid辅助因子存在于还原脱卤酶的厌氧菌，并检查各自的B12生物合成途径的变化。紫外/可见光谱，质谱分析和核磁共振技术将用于表征分离的corrinoids。基因组挖掘、蛋白质交联和相互作用研究将有助于鉴定参与PceA成熟的蛋白质。相应基因的诱变将允许这些蛋白质的功能分析及其在类咕啉生物合成和掺入PceA前体中的作用。本研究将利用亚细胞定位技术，阐明类可啉生物合成的膜相关步骤。该项目旨在拓宽限制性知识的生物合成和纳入corrinoid辅因子参与还原脱卤厌氧细菌。

Corrinoid cofactors are formed in a protein-assisted multistep biosynthetic pathway. A unique corrinoid, norpseudo-B12, is a cofactor of the tetrachloroethene (PCE) reductive dehalogenase (PceA) of the gram-negative anaerobe Sulfurospirillum multivorans. PceA purified from the gram-positive anaerobe Desulfitobacterium hafniense strain PCE-S harbors a set of different corrinoid cofactors. The major goals of this project are to identify and characterize the diverse corrinoid cofactors present in reductive dehalogenases of anaerobic bacteria and to examine variations in the respective B12-biosynthetic pathways. UV/Vis-spectroscopy, mass spectrometric analyses, and nuclear magnetic resonance techniques will be used to characterize the isolated corrinoids. Genome mining, protein cross-linking, and interaction studies will help to identify proteins involved in the maturation of PceA. Mutagenesis of the respective genes will allow for a functional analysis of these proteins and their role in corrinoid biosynthesis and incorporation into the PceA precursor. Using subcellular localization techniques the membrane associated steps of corrinoid biosynthesis will be unravelled in this study. The project is meant to broaden the restricted knowledge on the biosynthesis and incorporation of corrinoid cofactors involved in the reductive dehalogenation by anaerobic bacteria.