Functional characterization of a gene encoding a dual domain for uridine kinase and uracil phosphoribosyltransferase in Arabidopsis thaliana.
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-alpha-l-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/uprtl 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.
Islam
M Rafiqul
MR
Department of Biological Sciences, Seoul National University, Seoul 151-742, Republic of Korea.
Kim
Hoyeun
H
Kang
Shin-Wook
SW
Kim
Jung-Sup
JS
Jeong
Young-Min
YM
Hwang
Hyun-Ju
HJ
Lee
So-Young
SY
Woo
Je-Chang
JC
Kim
Sang-Gu
SG
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000235:genetics; D001483:Base Sequence; D017931:DNA Primers; D018744:DNA, Plant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D020224:Expressed Sequence Tags; D017930:Genes, Reporter; D008969:Molecular Sequence Data; D010430:Pentosyltransferases / Q000235:genetics*; D016415:Sequence Alignment; D014162:Transfection; D014541:Uridine Kinase / Q000235:genetics*
D029681:Arabidopsis Proteins; D017931:DNA Primers; D018744:DNA, Plant; D010430:Pentosyltransferases; C023269:uracil phosphoribosyltransferase; D014541:Uridine Kinase