Cloning, overexpression, and characterization of the functional dihydroorotase domain of the mammalian multifunctional protein CAD.
Mammalian dihydroorotase (DHOase) is part of a large multidomain protein called CAD, which initiates the first three steps in the de novo pyrimidine biosynthetic pathway. DHOase activity is carried out by a 44-kDa structural domain which could be isolated in active form from elastase digests. A core domain the same size as monofunctional dihydroorotases was defined, although the domain borders were uncertain. Two recombinants were overexpressed in Escherichia coli. The first encoded the core domain with 55 and 13 residues added to the amino and carboxyl ends, respectively, and was expressed in insoluble form. The recombinant protein was refolded from urea into a soluble form which was resistant to protease digestion but was catalytically inactive. In contrast, the proteolytic fragment from CAD could be unfolded and refolded with recovery of 40-100% catalytic activity. The second construct, which approximated the proteolytic fragment, had 21 residues on the amino end and 65 residues on the carboxyl end of the core domain. A 46-kDa soluble protein was expressed at 4% of the total soluble protein. The recombinant protein was catalytically active and had the expected amino-terminal sequence. The protein was purified to homogeneity. Dihydroorotase saturation curves gave a Km = 41.9 +/- 3.5 microM and a kcat = 2.79 +/- 0.06 s-1, parameters that were similar to those obtained for the proteolytic fragment. The Km was 6-fold higher and the kcat 2-fold lower than the values obtained for the parent protein, which suggests that interdomain interactions stabilize the active conformation.(ABSTRACT TRUNCATED AT 250 WORDS)
Zimmermann
B H
BH
Department of Biochemistry, Wayne State University School of Medicine, Detroit, Michigan 48201.
Evans
D R
DR
D000818:Animals; D001221:Aspartate Carbamoyltransferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D006224:Cricetinae; D004080:Dihydroorotase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D015151:Immunoblotting; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments / Q000302:isolation & purification; D010957:Plasmids; D016133:Polymerase Chain Reaction; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid
C033626:CAD trifunctional enzyme; D009097:Multienzyme Complexes; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D001221:Aspartate Carbamoyltransferase; D004080:Dihydroorotase; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)