Corrinoid cofactor biosynthesis and insertion into reductive dehalogenases of anaerobic bacteria

类角质辅因子是在蛋白质辅助的多步骤生物合成途径中形成的。一种独特的椰素，非伪b12，是革兰氏阴性厌氧菌多角硫螺旋菌的四氯乙烯（PCE）还原脱卤酶（PceA）的辅助因子。从革兰氏阳性厌氧菌hafniense Desulfitobacterium PCE-S中纯化的PceA含有一组不同的类角质辅因子。该项目的主要目标是识别和表征厌氧细菌还原脱卤酶中存在的各种类玉米粉辅助因子，并检查各自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被用来表征分离的角状体。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。对各自基因的诱变将允许对这些蛋白质进行功能分析，并分析它们在类椰碱生物合成和融入PceA前体中的作用。利用亚细胞定位技术，本研究将揭示类蓖麻素生物合成的膜相关步骤。该项目旨在拓宽有关厌氧细菌还原性脱卤过程中类椰碱辅助因子的生物合成和结合的有限知识。

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.