Cloning and nucleotide sequence of the urea amidolyase gene from Candida utilis

Abstract

Urea amidolyase (EC 3.5.1.45) is an important multi-functional enzyme for the degradation of urea. The urea amidolyase gene from Candida utilis CA(u)-37 ( DUR1,2 c ) was cloned by plaque hybridization, and the nucleotide sequences of DUR1, 2 c and its flanking regions were determined. DUR1, 2 c was found to be composed of 5,490 base pairs and 1,830 amino acid residues. Using Edman degradation of the purified enzyme, it was 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), since cDNA of DUR1, 2 c was synthesized from mRNA and sequenced. The nucleotide sequences of the urea amidolyase gene from Saccharomyces cerevisiae and DUR1, 2 c 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 enzyme. 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 the active site of allophanate hydrolase, that of urea carboxylase, and the biotin-binding site. This was verified by deletion analysis of the DUR1, 2 c gene in S. cerevisiae. The function of the upstream region of the C. utilis gene is also discussed.