Expression of catalytic domains of human UMP synthase in uridine auxotrophic bacteria.
Orotate phosphoribosyltransferase (OPRT) and orotidine-5'-monophosphate decarboxylase (ODC), which catalyze the last two steps in de novo UMP biosynthesis, are two distinct monofunctional proteins in bacteria and lower eukaryotes. In mammals, OPRT and ODC activities are contained in a single bifunctional protein labeled UMP synthase. The human UMP synthase cDNA was separated into the predicted OPRT and ODC domains using polymerase chain reaction techniques and the domains inserted into pUC19 expression vectors. Following transformation into OPRT- and ODC-deficient E. coli, the strains were able to grow on minimal media without uridine. The ODC-transformed bacteria expressed up to 24 times the level of activity found in a wild-type E. coli line. The OPRT-transformed E. coli contained only 4-9% of wild-type activity. Western blot analysis with antiserum to human UMP synthase demonstrates that OPRT and ODC domains are being produced in the deficient cells by the respective vectors. The level of the domain protein approximates the level of enzyme activity. The complementation of the OPRT and ODC activities in the transformed deficient E. coli strains demonstrates that human UMP synthase can be separated into active monofunctional domains that will function in the bacterial cell environment.
Department of Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101.
T
T
Lin
D P
DP
Suttle
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004926:Escherichia coli; D005816:Genetic Complementation Test; D006801:Humans; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism; D009962:Orotate Phosphoribosyltransferase / Q000235:genetics* / Q000378:metabolism; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000235:genetics* / Q000378:metabolism; D014529:Uridine / Q000378:metabolism*
D004269:DNA, Bacterial; D009097:Multienzyme Complexes; C027157:uridine 5'-monophosphate synthase; D009962:Orotate Phosphoribosyltransferase; D009964:Orotidine-5'-Phosphate Decarboxylase; D014529:Uridine