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

钼辅基是含钼酶中具有催化活性的辅基。莫科是深埋在其同源酶的内部。虽然已知一些细菌Mo-酶的分子伴侣负责Moco-插入，但尚未鉴定出真核同源物。因此，本项目的目的是鉴定Moco插入真核Mo-酶所必需的因子。由于我们已经达到了植物系统的技术极限，我们选择了粗糙脉孢菌作为更适合的系统，其中我们已经建立了一个完全定义的体外系统，以基于重组无Moco的脉孢菌硝酸还原酶测定Moco插入。我们的初步结果表明，Moco插入到脉孢菌硝酸还原酶是依赖于一个未知的因素。我们希望在分子和生物化学水平上描述这一因素。我们将产生和表征相应的敲除突变体，我们将在其他真核生物中鉴定该因子的同源物。我们希望确定参与血红素和FAD插入脉孢菌硝酸还原酶的其他因子，并揭示其插入序列。最后，我们想知道是否Moco插入到脉孢菌硝酸还原酶与脉孢菌氮代谢共调节。

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.