title: PYD2 encodes 5,6-dihydropyrimidine amidohydrolase, which participates in a novel fungal catabolic pathway.
abstract: 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 techniques have been modified to clone pyrimidine catabolic genes, study their expression and purify the corresponding enzymes from this yeast. The pyd2-1 mutant, which lacked the 5,6-dihydropyrimidine amidohydrolase (DHPase) activity, was transformed with wild-type S. kluyveri genomic library. The complementing 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-codons open reading frame was interrupted by a 63 bp intron, which does not contain the Saccharomyces cerevisiae branch-point sequence, and the transcripts contained a long 5' untranslated leader with five or six AUG codons. The derived amino acid sequence showed similarities with dihydroorotases, allantoinases 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 other non-fungal organisms. The S. kluyveri DHPase was purified to homogeneity and sequencing of the N-terminal region revealed that the purified enzyme corresponds to the PYD2 gene product. The enzyme is a tetramer, likely consisting 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 of the yeast PYD2 gene and DHPase seem to be different from that in other organisms. DHPase activity and Northern analysis demonstrated that PYD2 expression is inducible by dihydrouracil, though not by uracil. Apparently, dihydrouracil and DHPase represent an important regulatory checkpoint of the pyrimidine catabolic pathway in S. kluyveri.
authors.affiliation: Department of Microbiology Building 301, Technical University of Denmark, Lyngby, DK-2800, Denmark.
authors.forename: Z
authors.initials: Z
authors.lastname: Gojkovic
authors.forename: K
authors.initials: K
authors.lastname: Jahnke
authors.forename: K D
authors.initials: KD
authors.lastname: Schnackerz
authors.forename: J
authors.initials: J
authors.lastname: Piskur
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics; D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli; D019143:Evolution, Molecular; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012440:Saccharomyces / Q000201:enzymology* / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
chemical_list: D020121:5' Untranslated Regions; D011994:Recombinant Proteins; D000581:Amidohydrolases; C020047:dihydropyrimidinase