Poster: Enzymology

Abs # 237: Expression of a gene with dual domain for uracil phosphoribosyltransferase and uridine kinase from Arabidopsis thaliana leads to growth inhibition to 5-fluorouracil and 5-fluorouridine in Escherichia coli

Presenter:	Kim, Jung-Sup , dnabio@snu.ac.kr
Authors	Kim, Jung-Sup  (A)   Kang, Shin-Wook  (A)   Kim, Sang-Gu  (A)
Affiliations:	(A): Seoul National University

Uracil phosphoribosyltransferase (UPRT, EC 2.4.2.9) and uridine kinase (UDK, EC 2.7.1.48) are enzymes catalyzing the formation of uridine 5กฏ-monophosphate from uracil and 5-phosphoribosyl-1-pyrophosphate (PRPP) or urindine and adenosine 5กฏ-triphosphate (ATP) in pyrimidine salvage pathway, respectively. The genes for UPRT or UDK were reported in protozoa, bacteria, or yeasts as separate genes, but not in plants and animals. Here we report the characterization and functional analysis of a gene with dual function for UPRT and UDK from Arabidopsis thaliana (UKPP1). Sequencing of an EST clone was revealed that it contains a full-length open reading frame for UKPP1 which is 1461bp and encoded approximately 53kDa protein. The predicted amino acid sequence of UKPP1 is similar to the two protein family for UPRT and UDK. Carboxyl-terminal region is similar to upp of Escherichia coli and FUR1 of Saccharomyces cerevisiae and contains signature-binding motif for a uracil and a PRPP whereas the amino-terminal is similar to udk of E. coli and URK1 of S. cerevisiae and contains an ATP/GTP-binding site motif A called P-loop and putative substrate acceptor sites. Overexpression of UKPP1 in wild type and upp mutant of E. coli led to growth inhibition effect with 5-fluorouracil or 5-fluorouridine. These results suggest that the UKPP1 product can use uracil or uridine as a substrate and has UPRT and UDK enzyme activity.