Author/Editor		Gojković, Zoran; Jahnke, Karin; Schnackerz, Klaus D; Piškur, Jure
Title		PYD2 encodes 5,6-dihydropyrimidine amidohydrolase, which participates in a novel fungal catabolic pathway
Type		članek
Source		J Mol Biol
Vol. and No.		Letnik 295, št. 4
Publication year		2000
Volume		str. 1073-87
Language		eng
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. (Abstract truncated at 2000 characters)
Descriptors		AMIDOHYDROLASES AMINO ACID SEQUENCE ANIMALS BASE SEQUENCE CLONING, MOLECULAR ESCHERICHIA COLI EVOLUTION, MOLECULAR GENE EXPRESSION REGULATION, ENZYMOLOGIC MOLECULAR SEQUENCE DATA OPEN READING FRAMES PHYLOGENY RECOMBINANT PROTEINS SACCHAROMYCES SACCHAROMYCES CEREVISIAE SEQUENCE ALIGNMENT SEQUENCE HOMOLOGY, AMINO ACID