Class-II dihydroorotate dehydrogenases from three phylogenetically distant fungi support anaerobic pyrimidine biosynthesis

Abstract

Background In most fungi, quinone-dependent Class-II dihydroorotate dehydrogenases (DHODs) are essential for pyrimidine biosynthesis. Coupling of these Class-II DHODHs to mitochondrial respiration makes their in vivo activity dependent on oxygen availability. Saccharomyces cerevisiae and closely related yeast species harbor a cytosolic Class-I DHOD (Ura1) that uses fumarate as electron acceptor and thereby enables anaerobic pyrimidine synthesis. Here, we investigate DHODs from three fungi (the Neocallimastigomycete Anaeromyces robustus and the yeasts Schizosaccharomyces japonicus and Dekkera bruxellensis) that can grow anaerobically but, based on genome analysis, only harbor a Class-II DHOD. Results Heterologous expression of putative Class-II DHOD-encoding genes from fungi capable of anaerobic, pyrimidine-prototrophic growth (Arura9, SjURA9, DbURA9) in an S. cerevisiae ura1Δ strain supported aerobic as well as anaerobic pyrimidine prototrophy. A strain expressing DbURA9 showed delayed anaerobic growth without pyrimidine supplementation. Adapted faster growing DbURA9-expressing strains showed mutations in FUM1, which encodes fumarase. GFP-tagged SjUra9 and DbUra9 were localized to S. cerevisiae mitochondria, while ArUra9, whose sequence lacked a mitochondrial targeting sequence, was localized to the yeast cytosol. Experiments with cell extracts showed that ArUra9 used free FAD and FMN as electron acceptors. Expression of SjURA9 in S. cerevisiae reproducibly led to loss of respiratory competence and mitochondrial DNA. A cysteine residue (C265 in SjUra9) in the active sites of all three anaerobically active Ura9 orthologs was shown to be essential for anaerobic activity of SjUra9 but not of ArUra9. Conclusions Activity of fungal Class-II DHODs was long thought to be dependent on an active respiratory chain, which in most fungi requires the presence of oxygen. By heterologous expression experiments in S. cerevisiae, this study shows that phylogenetically distant fungi independently evolved Class-II dihydroorotate dehydrogenases that enable anaerobic pyrimidine biosynthesis. Further structure–function studies are required to understand the mechanistic basis for the anaerobic activity of Class-II DHODs and an observed loss of respiratory competence in S. cerevisiae strains expressing an anaerobically active DHOD from Sch. japonicus.

Identification of fungal dihydrouracil-oxidase genes by expression in Saccharomyces cerevisiae

“…2) was unexpected, since the only known fungal Class-I-A DHODs are proposed to have been obtained by horizontal gene transfer (Gojković et al 2004). Most of these sequences were found in proteomes of aerobic fungi that also contain a Class-II DHOD (Bouwknegt et al 2021). This observation raised the question whether and why these organisms harbour different, seemingly redundant DHODs.…”

“…This notion was first questioned when the facultatively anaerobic yeast Dekkera bruxellensis was shown to only contain a DHOD gene with sequence similarity to yeast Class-II DHOD genes (Woolfit et al 2007;Piškur et al 2012). We recently showed that expression of Class-II DHOD genes from D. bruxellensis, from obligately anaerobic Neocallimastigomycota, or from the facultatively anaerobic fission yeast Schizosaccharomyces japonicus, supported anaerobic growth of S. cerevisiae ura1Δ strains without pyrimidine supplementation (Bouwknegt et al 2021). These results indicated that acquisition of a Class-I DHOD by HGT is not the only mechanism by which fungi can evolve for anaerobic pyrimidine prototrophy.…”

“…a. In pyrimidine biosynthesis, fungal dihydroorotate dehydrogenase catalyses the oxidation of dihydroorotate orotate and donates electrons to the quinone pool of the respiratory chain (Class-II DHOD, but see(Bouwknegt et al 2021)) or to fumarate (Class-I DHOD). b.…”

Correction to: Class‑II dihydroorotate dehydrogenases from three phylogenetically distant fungi support anaerobic pyrimidine biosynthesis

“…Following publication of the original article [ 1 ], the authors reported errors in the text of the Results section and in Table 2. It refers to a mutation in a yeast gene as VPS1 I410L and to the corresponding change in the Vps1 amino-acid sequence as I410L.…”

Optimization of energy production and central carbon metabolism in a non-respiring eukaryote

“…The fission yeast Schizosaccharomyces japonicus (S. japonicus), together with the more widely used relative S. pombe, provide an attractive composite model system to study this question. Unlike S. pombe, S. japonicus thrives both in the presence and the absence of oxygen [22][23][24][25][26][27]. Despite encoding most genes required for respiration, it does not produce coenzyme Q, does not grow on a non-fermentable carbon source glycerol, and does not consume oxygen during vegetative growth in glucose, unlike its sister species [22,23,26,28].…”