Cloning and expression of the mammalian multifunctional protein CAD in Escherichia coli. Characterization of the recombinant protein and a deletion mutant lacking the major interdomain linker.
The multifunctional protein CAD catalyzes the first three steps in de novo pyrimidine biosynthesis in mammalian cells. Glutamine-dependent carbamyl-phosphate synthetase (CPSase), aspartate transcarbamylase, and dihydroorotase activities are carried by a 243-kDa polypeptide chain that is organized into discrete functional domains connected by interdomain linkers. One of the connecting chain segments, the DA linker bridging the dihydroorotase and aspartate transcarbamylase domains, is unusually long (109 residues) and conserved in length in all eukaryotic species. A plasmid (pCK-CAD10) that encodes the entire 243-kDa polypeptide was constructed and expressed in Escherichia coli. The recombinant protein was purified to homogeneity by ion exchange and gel filtration chromatography. The purified protein had kinetic parameters that were close to those obtained for native CAD. Moreover, the CPSase activity was allosterically regulated. Gel filtration showed that the recombinant protein had the same molecular mass as native CAD. Thus, this complex mammalian protein is expressed and folds correctly in bacterial cells and, despite its extreme protease sensitivity, can be isolated intact. A deletion mutant that lacked the DA linker was then constructed. The kinetic parameters of the mutant protein were, for the most part, unaltered, showing that the DA linker is not essential for the proper folding or optimal functioning of the individual domains. However, a significant decrease in the thermal stability of the CPSase domain suggested that the linker helps to stabilize the complex. Moreover, the channeling of carbamyl phosphate, determined by measuring the extent to which the exogenously added intermediate could dilute the endogenous carbamyl phosphate pool, was appreciably reduced when the DA linker was removed. Thus, although the domains function autonomously, some of the linkers are important for interdomain interactions in CAD.
Guy
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HI
Department of Biochemistry, Wayne State University School of Medicine, Detroit, Michigan 48201.
Evans
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DR
D000818:Animals; D001221:Aspartate Carbamoyltransferase / Q000235:genetics* / Q000378:metabolism; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000235:genetics* / Q000378:metabolism; D002221:Carbamyl Phosphate / Q000378:metabolism; D003001:Cloning, Molecular; D006224:Cricetinae; D004080:Dihydroorotase / Q000235:genetics* / Q000378:metabolism; D006358:Hot Temperature; D007700:Kinetics; D008647:Mesocricetus; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D013329:Structure-Activity Relationship
C033626:CAD trifunctional enzyme; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D002221:Carbamyl Phosphate; D001221:Aspartate Carbamoyltransferase; D004080:Dihydroorotase; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)