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

钼辅因子（Moco）是含钼酶中具有催化活性的辅基。在目前研究的所有真核生物中，Moco生物合成的第一步在线粒体中进行，而随后的所有步骤都在细胞质中进行。这一步骤由两种蛋白质催化，其中第一个蛋白质（niti - 7a）含有两个[4Fe-4S]型的FeS簇。但GTP作为起始化合物也存在于细胞质溶胶中。胞质溶胶中也有FeS簇合成机制。那么问题来了，为什么Moco生物合成步骤1位于线粒体中。以丝状真菌粗神经孢子菌为酵母样模型系统，分析了moco生物合成与FeS代谢之间的相互作用。在酵母（酿酒酵母）中，真核FeS簇生的所有原理都已被成功地研究出来，但酵母没有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。在酵母中成功表达nit7将开辟一个全新的视角，因为酵母FeS生物发生研究工具的完整库将在我们手中。显然，这一尝试还将揭示酵母线粒体出口蛋白Atm1p是否也能够出口Moco生物合成步骤1的产物，这是我们对其植物同源物ATM3的假设。Atm1p促进FeS簇等效物的出口。对于第二个项目线，在初步实验中，我们已经在nit-7敲除背景下在N. crassa细胞质中稳定地表达了Nit-7A。我们计划对该菌株进行如下表征：(1)表型（在硝酸盐培养基上生长产生对Moco和FeS的强烈需求），(2)生化（重新分离异位表达的nti - 7a和nti - 7ab并分析FeS簇），以及(3)转录组分析（硝酸盐诱导的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nti - 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？真菌和人类第一步Moco生物合成有什么不同？为了实现这一建议的目标，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.