Synthesis of the nonconserved dihydroorotase domain of the multifunctional hamster CAD protein in Escherichia coli.
CAD is the multifunctional protein of higher eukaryotes which catalyzes the first three steps of pyrimidine biosynthesis. Its enzymatic activities exist as independent domains in the order: N terminus-carbamylphosphate synthetase II(CPSase)-dihydroorotase(DHOase)-aspartate transcarbamylase(ATCase)-C terminus. To functionally define the minimum hamster cDNA region required to encode an active DHOase, expression constructs were generated. Many such constructs complement Escherichia coli mutants defective not only in DHOase but also in ATCase. Constructs deleted for most of the sequence encoding the ATCase domain continue to complement E. coli mutants defective in DHOase. All of these smaller constructs also lack the region encoding CPSase. Therefore, a 'genetic cassette', containing information for neither the CPSase nor the ATCase domain, can direct the synthesis of a polypeptide with DHOase activity. Interestingly, inclusion of a portion of the DHOase-ATCase interdomain bridge appears to be required for optimum activity.
Department of Microbiology and Immunology, Albert B. Chandler Medical Center, University of Kentucky, Lexington 40536-0084.
L A
LA
Musmanno
J A
JA
Maley
J N
JN
Davidson
D000595:Amino Acid Sequence; D000818:Animals; D001221:Aspartate Carbamoyltransferase / Q000235:genetics / Q000502:physiology; D001483:Base Sequence; D006224:Cricetinae / Q000235:genetics*; D004274:DNA, Recombinant; D004080:Dihydroorotase / Q000096:biosynthesis / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D014170:Transformation, Genetic
D004274:DNA, Recombinant; D001221:Aspartate Carbamoyltransferase; D004080:Dihydroorotase