Identification and characterization of human uracil phosphoribosyltransferase (UPRTase).
Uracil phosphoribosyltransferase, which catalyzes the conversion of uracil and 5-phosphoribosyl-1-R-diphosphate to uridine monophosphate, is important in the pyrimidine salvage pathway and is an attractive target for rational drug design by incorporation of prodrugs that are lethal to many parasitic organisms specifically. So far, uracil phosphoribosyltransferase has been reported in Arabidopsis thaliana only, not in mammals. In this study, a novel uracil phosphoribosyltransferase family cDNA encoding a 309 amino acid protein with a putative uracil phosphoribosyltransferase domain was isolated from the human fetal brain library. It was named human UPRTase (uracil phosphoribosyltransferase). The ORF of human UPRTase gene was cloned into pQE30 and expressed in Escherichia coli M15. The protein was purified by Ni-NTA affinity chromatography, but UPRTase activity could not be detected by spectrophotometry. RT-PCR analysis showed that human UPRTase was strongly expressed in blood leukocytes, liver, spleen, and thymus, with lower levels of expression in the prostate, heart, brain, lung, and skeletal muscle. Subcellular location of UPRTase-EGFP fusion protein revealed that human UPRTase was distributed in the nucleus and cytoplasm of AD293 cells. Evolutional tree analyses of UPRTases or UPRTase-domain-containing proteins showed that UPRTase was conserved in organisms. UPRTases of archaebacteria or eubacterium had UPRTase activity whereas those higher than Caenorhabditis elegans, which lacked two amino acids in the uracil-binding region, had no UPRTase activity. This means that human UPRTase may have enzymatic activity with another, unknown, factor or have other activity in pyrimidine metabolism.
Li
Jixi
J
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai, 200433, People's Republic of China.|Division of Biology, California Institute of Technology, Pasadena, CA, 91125, USA.
Huang
Shengdong
S
Department of Cardiac-thoracic Surgery, Changhai Hospital, Second Military Medical University, Shanghai, 200433, People's Republic of China.
Chen
Jinzhong
J
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai, 200433, People's Republic of China.
Yang
Zhenxing
Z
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai, 200433, People's Republic of China.
Fei
Xiangwei
X
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai, 200433, People's Republic of China.
Zheng
Mei
M
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai, 200433, People's Republic of China.
Ji
Chaoneng
C
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai, 200433, People's Republic of China. chnji@fudan.edu.cn.
Xie
Yi
Y
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai, 200433, People's Republic of China.
Mao
Yumin
Y
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai, 200433, People's Republic of China.
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary; D019143:Evolution, Molecular; D005333:Fetus / Q000378:metabolism; D015870:Gene Expression; D015723:Gene Library; D006801:Humans; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010430:Pentosyltransferases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D016415:Sequence Alignment
D018076:DNA, Complementary; D010430:Pentosyltransferases; C023269:uracil phosphoribosyltransferase