Structural and transcriptional analysis of the pyrABCN, pyrD and pyrF genes in Aspergillus nidulans and the evolutionary origin of fungal dihydroorotases

The six biochemical steps of the de novo pyrimidine biosynthesis pathway ar e conserved in all known organisms, However, in animals and fungi, unlike p rokaryotes, at least the first two activities are grouped on a multifunctio nal enzyme. Here, we report cloning, mapping and transcriptional characteri zation of some pyrimidine biosynthesis genes in the filamentous fungus Aspe rgillus nidulans. The first two steps of the pathway are performed by a mul tifunctional enzyme comprising the activities of carbamoyl phosphate synthe tase (CPSase) and aspartate transcar bamylase (ATCase), This polypeptide is encoded by a 7 kbp cluster gene, pyrABCN, which has a high degree of nucle otide identity with the Ura2 gene in Saccharomyces cerevisiae. The enzyme o f the third step, dihydroorotase (DHOase), is encoded by a separate locus, pyrD. However, the pyrABCN gene apparently contains an evolutionary remnant of a DHOase-encoding sequence, similarly to the Ura2 gene of Saccharomyces cerevisiae, The pyrABCN gene is transcribed as a single 7 kb mRNA species. The level of transcripts of pyrABCN, pyrD and, to a lesser degree, pyrF ge nes responds to the presence of exogenous pyrimidines and to the conditions of pyrimidine starvation. Derepression of pyrABCN and pyrD under pyrimidin e starvation is noticeably enhanced in pyrE mutants that accumulate dihydro orotic acid. The pyrABCN gene maps to the distal portion of the right arm o f the chromosome VIII, whereas the pyrD gene, in contrast to early genetic data, is closely linked to the brIA gene and located to the right of it. Ou r data on mitotic recombination should help to verify the genetic map of th e chromosome VIII. Comparison of amino acid sequences of active dihydroorot ases with related enzymes and with their non-functional homologues in yeast and Aspergillus indicates that the active dihydroorotases from fungi are m ore similar to ureases and enzymes of the pyrimidine degradation pathway. T he 'silent' dihydroorotase domains of the multifunctional enzymes from fung i and active DHOase domains of the multifunctional enzymes in higher eukary otes are more closely related to bacterial dehydroorotases.