Functional characterization of a gene encoding a dual domain for uridine kinase and uracil phosphoribosyltransferase in Arabidopsis thaliana

• Published in: Plant molecular biology Vol. 63; no. 4; pp. 465 - 477
• Main Authors: Islam, M. Rafiqul, Kim, Hoyeun, Kang, Shin-Wook, Kim, Jung-Sup, Jeong, Young-Min, Hwang, Hyun-Ju, Lee, So-Young, Woo, Je-Chang, Kim, Sang-Gu
• Format: Journal Article
• Language: English
• Published: Netherlands Dordrecht : Kluwer Academic Publishers 01-03-2007; Springer Nature B.V
• Subjects: 5-Fluorouracil; 5-Fluorouridine; Amino Acid Sequence; Arabidopsis - enzymology; Arabidopsis - genetics; Arabidopsis Proteins - genetics; Arabidopsis thaliana; Bacteria; Base Sequence; DNA Primers; DNA, Plant - genetics; E coli; Escherichia coli; Escherichia coli - genetics; Expressed Sequence Tags; Genes, Reporter; Molecular Sequence Data; Mutants; Pentosyltransferases - genetics; Proteins; Sequence Alignment; Transfection; UMP synthesis; Uracil phosphoribosyltransferase; Uridine kinase; Uridine Kinase - genetics
• ISSN: 0167-4412; 1573-5028
• DOI: 10.1007/s11103-006-9101-3

Uridine kinase (UK) and uracil phosphoribosyltransferase (UPRT) are enzymes catalyzing the formation of uridine 5'-monophosphate (UMP) from uridine and adenine 5'-triphosphate (ATP) and from uracil and phosphoribosyl-α-1-pyrophosphate (PRPP), respectively, in the pyrimidine salvage pathway. Here, we report the characterization and functional analysis of a gene AtUK/UPRT1 from Arabidopsis thaliana. Sequencing of an expressed sequence tag clone of this gene revealed that it contains a full-length open reading frame of 1461 nucleotides and encodes a protein with a molecular mass of approximately 53 kDa. The sequence analysis revealed that the N-terminal region of AtUK/UPRT1 contains a UK domain and the C-terminal region consists of a UPRT domain. Expression of AtUK/UPRT1 in upp and upp-udk mutants of Escherichia coli supplied with 5-fluorouracil (5-FU) and 5-fluorouridine (5-FD) led to growth inhibition. Identical results were obtained with 5-FD and 5-FU treatments when the UK and UPRT domains were separated by the introduction of translation initiation and stop codons prior to complementation into the upp-udk and upp mutants. These results suggest that the AtUK/UPRT1 product can use uracil and uridine as substrates for the production of UMP. We also investigated the function of AtUK/UPRT1 in an Arabidopsis mutant. The wild-type Arabidopsis plants showed drastic growth retardation when they were treated with 5-FU and 5-FD while the growth of atuk/uprt1 mutant plants was not significantly affected. These findings confirm that AtUK/UPRT1 has a dual role in coding for both uridine kinase and uracil phosphoribosyltransferase that form UMP through the pyrimidine salvage pathway in Arabidopsis.