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）是含钼酶中的催化活性辅基。在迄今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中，而所有后续步骤都在细胞质中进行。该步骤由两种蛋白质催化，其中第一个蛋白质 (Nit-7A) 含有两个 [4Fe-4S] 类型的 FeS 簇。但 GTP 作为起始化合物也可在细胞质中获得。细胞质还含有 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。我们希望使用丝状真菌粗糙脉孢菌作为类酵母模型系统来分析 Moco 生物合成和 FeS 代谢之间的串扰。在酵母（酿酒酵母）中，真核 FeS 簇生物发生的所有原理均已成功阐明，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。 nit-7 在酵母中的成功表达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掌握在我们手中。显然，这一尝试还可以揭示促进 FeS 簇等价物输出的酵母线粒体输出蛋白 Atm1p 是否也能够输出 Moco 生物合成步骤 1 的产物，我们假设其植物同源物 ATM3。对于第二个项目系，在初步实验中，我们已经在 nit-7 敲除背景下在粗糙链孢菌的细胞质中异位且稳定地表达 Nit-7A。我们计划通过（i）表型（硝酸盐培养基上的生长对 Moco 产生强烈需求，从而对 FeS 产生强烈需求）、（ii）生物化学（异位表达的 Nit-7A 和 Nit-7AB 的重新分离以及 FeS 簇的分析）和（iii）通过转录组分析（FeS 簇组装和 Moco 生物合成中硝酸盐诱导的转录变化）来表征，以解决以下问题：是细胞质中异位表达的 Nit-7A 的 FeS 簇的供体吗？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 1 步 Moco 生物合成有差异吗？为了实现该提案的目标，已经准备好与优先计划内的四个小组进行密切合作。

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.