PYD2 encodes 5,6-dihydropyrimidine amidohydrolase, which participates in anovel fungal catabolic pathway

Most fungi cannot use pyrimidines or their degradation products as the sole nitrogen source. Previously, we screened several yeasts for their ability to catabolise pyrimidines. One of them, Saccharomyces kluyveri, was able to degrade the majority of pyrimidines. Here, a series of molecular technique s have been modified to clone pyrimidine catabolic genes, study their expre ssion and purify the corresponding enzymes from this yeast. The pyd2-1 muta nt which lacked the 5,6-dihydropyrimidine amidohydrolase (DHPase) activity, was transformed with wild-type S. kluyveri genomic library. The complement ing plasmid contained the full sequence of the PYD2 gene, which exhibited a high level of homology with mammalian DHPases and bacterial hydantoinases. The organisation of PYD2 showed a couple of specific features. The 542-cod ons open reading frame was interrupted by a 63 bp intron, which does not co ntain the Saccharomyces cerevisiae branch-point sequence, and the transcrip ts contained a long 5' untranslated leader with five or six AUG codons. The derived amino acid sequence showed similarities with dihydroorotases, alla ntoinases and uricases from various organisms. Surprisingly, the URA4 gene from S. cerevisiae, which encodes dihydroorotase, shows greater similarity to PYD2 and other catabolic enzymes than to dihydroorotases from several ot her non-fungal organisms. The S. kluyveri DHPase was purified to homogeneit y and sequencing of the N-terminal region revealed that the purified enzyme corresponds to the PYD2 gene product. The enzyme is a tetramer, likely con sisting of similar if not identical subunits each With a molecular mass of 59 kDa. The S. kluyveri DHPase was capable of catalysing both dihydrouracil and dihydrothymine degradation, presumably by the same reaction mechanism as that described for mammalian DHPase. On the other hand, the regulation o f the yeast PYD2 gene and DHPase seem to be different from that in other or ganisms. DHPase activity and Northern analysis demonstrated that PYD2 expre ssion is inducible by dihydrouracil, though not by uracil. Apparently, dihy drouracil and DHPase represent an important regulatory checkpoint of the py rimidine catabolic pathway in S. kluyveri. (C) 2000 Academic Press.