Bouwknegt Jonna, Koster Charlotte C, Vos Aurin M, Ortiz-Merino Raúl A, Wassink Mats, Luttik Marijke A H, van den Broek Marcel, Hagedoorn Peter L, Pronk Jack T
Department of Biotechnology, Delft University of Technology, van der Maasweg 9, 2629 HZ, Delft, The Netherlands.
Wageningen Plant Research, Wageningen University and Research, Droevendaalsesteeg 1, 6708 PB, Wageningen, The Netherlands.
Fungal Biol Biotechnol. 2021 Oct 16;8(1):10. doi: 10.1186/s40694-021-00117-4.
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.
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.
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.
在大多数真菌中，醌依赖性II类二氢乳清酸脱氢酶（DHODs）对于嘧啶生物合成至关重要。这些II类DHODHs与线粒体呼吸的偶联使得它们的体内活性依赖于氧气供应。酿酒酵母和密切相关的酵母物种含有一种胞质I类DHOD（Ura1），它使用富马酸作为电子受体，从而实现厌氧嘧啶合成。在这里，我们研究了三种能够厌氧生长但基于基因组分析仅含有II类DHOD的真菌（新美鞭菌纲的粗壮厌氧霉菌以及酵母日本裂殖酵母和布鲁塞尔德克酵母）中的DHODs。
在酿酒酵母ura1Δ菌株中，对能够厌氧、嘧啶原养型生长的真菌（Arura9、SjURA9、DbURA9）中假定的II类DHOD编码基因进行异源表达，支持有氧以及厌氧嘧啶原养型。表达DbURA9的菌株在不补充嘧啶的情况下厌氧生长延迟。适应后生长更快的表达DbURA9的菌株在编码延胡索酸酶的FUM1中出现突变。绿色荧光蛋白标记的SjUra9和DbUra9定位于酿酒酵母线粒体，而序列中缺乏线粒体靶向序列的ArUra9定位于酵母细胞质。细胞提取物实验表明，ArUra9使用游离的FAD和FMN作为电子受体。在酿酒酵母中表达SjURA9可重复性地导致呼吸能力丧失和线粒体DNA丢失。所有三种具有厌氧活性的Ura9直系同源物活性位点中的一个半胱氨酸残基（SjUra9中的C265）被证明对于SjUra9的厌氧活性至关重要，但对ArUra9并非如此。
长期以来人们一直认为真菌II类DHODs的活性依赖于活跃的呼吸链，而在大多数真菌中这需要氧气的存在。通过在酿酒酵母中的异源表达实验，本研究表明系统发育距离较远的真菌独立进化出了能够进行厌氧嘧啶生物合成的II类二氢乳清酸脱氢酶。需要进一步的结构 - 功能研究来理解II类DHODs厌氧活性的机制基础以及在表达来自日本裂殖酵母的具有厌氧活性DHOD的酿酒酵母菌株中观察到的呼吸能力丧失现象。
