Biochemical and molecular studies of molybdenum cofactor-insertion into nitrate reductase of Neurospora crassa

点击翻译按钮获取中文摘要

Molybdenum cofactor (Moco) is the catalytically active prosthetic group in Mo-containing enyzmes. Moco is deeply buried within the interior of its cognate enzymes. While for some bacterial Mo-enzymes chaperons responsible for Moco-insertion are known, no eukaryotic homologs have been identified yet. Therefore, the aim of this project is to identify factor(s) that are necessary for Moco-insertion into eukaryotic Mo-enzymes. As we have reached the technical limits of the plant system, we chose Neuropsora crassa as better suited system where we have established a fully defined in vitro-system to assay for Moco-insertion, based on recombinant Moco-free Neurospora nitrate reductase. Our preliminary results showed that Moco-insertion into Neurospora nitrate reductase is dependent on a yet unknown factor. We wish to characterize this factor on the molecular and biochemical level. We will generate and characterize the corresponding knock-out mutant, and we will identify homologs to this factor in other eukaryotes. We wish to identify additional factors that are involved in the insertion of heme and FAD into Neurospora nitrate reductase and unveil the sequence of their insertion. Finally, we want to find out if Moco-insertion into Neurospora nitrate reductase is co-regulated with Neurospora nitrogen metabolism.