The aspartate transcarbamylase domain of a mammalian multifunctional protein expressed as an independent enzyme in Escherichia coli.
Although aspartate transcarbamylase (ATCase) is an independent, monofunctional enzyme in Escherichia coli, mammalian ATCase is one of the globular enzymatic domains of the multifunctional CAD protein. We subcloned fragments of the hamster CAD cDNA and assayed polypeptide products expressed in E. coli for ATCase activity in order to isolate a stretch of cDNA which encodes only the ATCase domain. Three such expression constructs contain fragments of hamster CAD cDNA similar in length to the gene encoding the E. coli ATCase catalytic subunit (pyrB). These constructs yield stable proteins with ATCase activity, ascertained by both in vivo and in vitro assays; the clones also possess sequence homology with the pyrB gene at both the 5' and 3' ends. The clone producing the most active ATCase contains cDNA which is analogous to the entire pyrB gene, plus a small amount of CAD sequence upstream of this region. Because these constructs produce independently folded, active ATCase from a piece of cDNA the size of the E. coli pyrB gene, they open the door for the in-depth investigation of the isolated mammalian enzyme domain utilizing recombinant DNA technology. This approach is potentially useful for the analysis of domains of other multifunctional proteins.
D000581:Amidohydrolases / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001221:Aspartate Carbamoyltransferase / Q000235:genetics*; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000235:genetics*; D003001:Cloning, Molecular; D006224:Cricetinae; D004247:DNA / Q000235:genetics; D004080:Dihydroorotase / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics*; D010957:Plasmids
C033626:CAD trifunctional enzyme; D009097:Multienzyme Complexes; D004247:DNA; D001221:Aspartate Carbamoyltransferase; D000581:Amidohydrolases; D004080:Dihydroorotase; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)