Molybdenum cofactor-biosynthesis and crosstalk to FeS metabolism in Neurospora crassa after ectopic expression of Moco biosynthesis step 1 proteins

钼辅基(MoCo)是含钼酶中具有催化活性的辅基。在到目前为止研究的所有真核生物中，MOCO生物合成的第一步位于线粒体，而所有后续步骤都在胞浆中进行。这一步骤是由两种蛋白质催化的，其中第一种蛋白质(nit-7A)含有两个[4Fe-4S]型FeS簇。但GTP作为起始化合物也存在于细胞质中。胞浆中还含有FeS簇合成装置。因此，出现了一个问题，为什么MOCO生物合成步骤1位于线粒体中。以丝状真菌粗糙脉孢霉作为酵母类模型系统，分析微生物生物合成与FeS代谢之间的串扰。在酵母(S.cerevisiae)中，真核生物FeS簇生物发生的所有原理都已被成功地阐明，但酵母中没有Mo代谢。在第一个项目中，我们计划在酵母中表达n.crassa nit-7基因，从而在酵母中建立MOCO生物合成步骤1。NIT-7在酵母中的成功表达将打开一个全新的视角，因为完整的酵母FeS生物发生研究工具将掌握在我们手中。显然，这一尝试也将允许揭示促进FeS簇等效物出口的酵母线粒体出口商Atm1p是否也能够出口MOCO生物合成步骤1的产物，我们假设这是其植物同源物ATM3。对于第二个项目路线，在初步实验中，我们已经在nit-7敲除背景下异位稳定地在粗劣新月球藻的胞浆中表达了nit-7A。我们计划鉴定这株菌株的表型(I)表型(在硝酸盐介质上生长产生对MOCO的强烈需求，因此对FES也有强烈的需求)，(Ii)生物化学(重新分离异位表达的NIT-7A和NIT-7AB并分析FES-簇)，以及(Iii)通过转录组分析(硝酸盐诱导FES簇组装和MOCO生物合成的转录变化)，以解决以下问题：细胞质中异位表达的NIT-7A的FES簇的供体是什么？这个过程是否与MoCO-和FeS-生物合成共同调节？真菌和人类的第一步MOCO生物合成有区别吗？为实现这项建议的目标，已准备与优先方案内的四个小组密切合作。

Molybdenum cofactor (Moco) is the catalytically active prosthetic group in Mo-containing enyzmes. In all eukaryotes studied so far, the first step of Moco biosynthesis resides in the mitochondria while all subsequent steps proceed in the cytosol. This step is catalyzed by two proteins where the first one (Nit-7A) harbors two FeS clusters of the [4Fe-4S] type. But GTP as starting compound is also available in the cytosol. And the cytosol harbors also a FeS cluster synthesis machinery. So the question arises why Moco biosynthesis step 1 is located in the mitochondria. Using the filamentous fungus Neurospora crassa as yeast-like model system we wish to analyse the crosstalk between Moco-biosynthesis and FeS metabolism. In yeast (S. cerevisiae) all principles of eukaryotic FeS cluster biogenesis have been successfully worked out, but yeast has no Mo metabolism. In a first project line, we plan to express the N. crassa nit-7 gene in yeast thus establishing Moco biosynthesis step 1 in yeast. A successful expression of nit-7 in yeast would open up a whole new perspective because the complete arsenal of yeast FeS biogenesis research tools would be at our hands. Clearly, this attempt would also allow to uncover whether the yeast mitochondrial exporter Atm1p which facilitates the export of FeS cluster equivalents, would be able to export also the product of Moco biosynthesis step 1, which we have postulated for its plant homolog ATM3. For a second project line, in preliminary experiments we have already expressed Nit-7A ectopically and stably in the cytosol of N. crassa in a nit-7 knock out-background. We plan to characterize this strain (i) phenotypically (growth on nitrate-media creates a strong demand for Moco and thus also for FeS), (ii) biochemically (re-isolation of ectopically expressed Nit-7A and Nit-7AB and analysis of FeS-clusters), and (iii) via transcriptome analysis (nitrate-induced transcriptional changes in FeS cluster assembly and Moco biosynthesis) in order to address the following questions: What is the donor of FeS clusters for ectopically expressed Nit-7A in the cytosol? Is this process co-regulated with Moco- and FeS-biosynthesis? Are there differences between fungal and human step 1 Moco biosynthsis? To meet the objectives of this proposal, a tight collaboration with four groups within the Priority Program has been prepared.