Expression and sequence analysis of a cDNA encoding the orotidine-5'-monophosphate decarboxylase domain from Ehrlich ascites uridylate synthase.
Orotidine-5'-monophosphate decarboxylase (OD-Case) catalyzes the conversion of orotidine 5'-monophosphate to UMP. In mammals, ODCase is present as part of a bifunctional protein which also contains orotate phosphoribosyltransferase; the preceding enzyme in the de novo UMP biosynthetic pathway. We have isolated a plasmid (pMEJ) which contains a cDNA for the ODCase domain of UMP synthase. Insertion of this sequence into an Escherichia coli expression vector (pUC12) has allowed for the expression of ODCase and not orotate phosphoribosyltransferase in E. coli. The molecular weight of the expressed protein is 26,000-27,300 from immunoblot analysis which corresponds closely to the molecular weight of the ODCase domain (28,500) isolated by tryptic digestion of UMP synthase. We have sequenced the cDNA insert of pMEJ and deduced the amino acid sequence. The molecular weight of the ODCase domain calculated from the amino acid sequence in 28,654. Comparison of the deduced amino acid sequence from pMEJ with that for yeast ODCase (a monofunctional protein) demonstrated that 52% of the amino acids were identical when the two sequences are compared. Furthermore, several stretches of the amino acid sequence have 80% or greater absolute homology.
C A
CA
Ohmstede
S D
SD
Langdon
C B
CB
Chae
M E
ME
Jones
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002262:Carboxy-Lyases / Q000235:genetics*; D002286:Carcinoma, Ehrlich Tumor / Q000201:enzymology*; D004247:DNA / Q000032:analysis*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005786:Gene Expression Regulation; D007163:Immunosorbent Techniques; D009097:Multienzyme Complexes / Q000235:genetics*; D009962:Orotate Phosphoribosyltransferase / Q000235:genetics*; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000235:genetics*; D010430:Pentosyltransferases / Q000235:genetics*; D010957:Plasmids; D011061:Poly A / Q000302:isolation & purification; D012313:RNA / Q000302:isolation & purification; D012333:RNA, Messenger; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics
D009097:Multienzyme Complexes; D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; C027157:uridine 5'-monophosphate synthase; D004247:DNA; D010430:Pentosyltransferases; D009962:Orotate Phosphoribosyltransferase; D004262:DNA Restriction Enzymes; D002262:Carboxy-Lyases; D009964:Orotidine-5'-Phosphate Decarboxylase