CLONING AND NUCLEOTIDE-SEQUENCE OF THE UREA AMIDOLYASE GENE FROM CANDIDA-UTILIS

Urea amidolyase (EC 3.5.1.45) is an important multi-functional enzyme for the degradation of urea. The urea amidolyase gene from Candida uti lis CA(u)-37 (DUR1, 2C) was cloned by plaque hybridization, and the nu cleotide sequences of DUR1, 2C and its flanking regions were determine d. DUR1, 2C was found to be composed of 5,490 base pairs and 1,830 ami no acid residues. Using Edman degradation of the purified enzyme, it w as revealed that the amino-terminal residue (methionine) was processed for maturation. A TATA-box like sequence was found 112 bases upstream from the translation start site (ATG). The site of the poly (A) tail was found 54 bases downstream from the translation stop site (TGA), si nce cDNA of DUR1, 2C was synthesized from mRNA and sequenced. The nucl eotide sequences of the urea amidolyase gene from Saccharomyces cerevi siae and DUR1, 2C were very similar to each other (65.3%), as were the deduced amino acid sequences (67.2%). The molecular weight of DUR1, 2 C was calculated to be 200,700. This value corresponded to the result obtained from SDS-polyacrylamide gel electrophoresis of the purified e nzyme. The enzyme functions in a dimeric form. Three important regions were found in the amino acid sequence of urea amidolyase through the homology search. It was predicted that each region was equivalent to t he active site of allophanate hydrolase, that of urea carboxylase, and the biotin-binding site. This was verified by deletion analysis of th e DUR1, 2C gene in S. cerevisiae. The function of the upstream region of the C. utilis gene is also discussed.