Insights into the regulation and post-transcriptional modifications of the molybdenum cofactor biosynthesis in Neurospora crassa

Abstract

The transition metal molybdenum (Mo) is present in all kingdoms of life, bound to vitally crucial enzymes. Mo itself is biologically inactive and needs a scaffold to serve as part of the active site of its user enzymes. This scaffold is a pyranopterin compound called molybdopterin (MPT). The four steps of MPT synthesis and Mo insertion, leading to the final pathway product molybdenum cofactor (Moco), are highly conserved. Moco plays an essential role in nitrate assimilation by binding to the active site of nitrate reductase (NR), the first and rate-limiting enzyme of this pathway. While the literature describes the regulatory mechanisms of nitrogen catabolism in detail, the knowledge about eukaryotic Moco biosynthesis regulation is limited. The focus of this work was to give first insights into the mechanisms leading to optimal Moco biosynthesis activity under Moco demanding conditions. For this, I used the fungal model organism Neurospora crassa and revealed a tight connection between enhanced NR expression and the Moco biosynthesis rate. The transcript quantification of genes involved in Moco biosynthesis showed that nit-7, encoding for the first step of Moco biosynthesis, is the critical point of regulation. While Moco demand caused the up-regulation of non-spliced nit-7 expression (encoding for NIT-7A), I saw no regulation of the splice variant encoding for NIT-7AB. The mRNA analysis identified a third, yet unknown splice variant, also encoding for NIT-7A. Following these findings, I examined the interplay of these variants, their encoded enzymes, and potential new protein products in different in vivo experiments. Mutagenesis and rescue studies showed that the NIT-7 A- and B-domain together catalyze the conversion of guanosine-5’-triphosphate to cyclic pyranopterin monophosphate (cPMP). NIT-7A harbors the sole mitochondrial targeting signal, and thus, NIT-7B requires the fusion to an inactive A-domain for its translocation. After import, the mitochondria separate the domains of NIT-7AB and degrade the A-domain. Not only fungi but also animals exhibit the fusion of A- and B-domain of NIT-7 orthologs. The precise analysis of the NIT-7 import identified that the opisthokont-branch developed at least two different strategies to orchestrate the cytoplasmic export of fused cPMP-synthase enzymes.