Expression of catalytically active hamster dihydroorotase domain in Escherichia coli: purification and characterization.
Dihydroorotase is the central domain of trifunctional L-dihydroorotate synthetase which also contains carbamyl phosphate synthetase at the N-terminus and aspartate transcarbamylase at the C-terminus. The cDNA, corresponding to the active dihydroorotase domain as isolated after digestion of dihydroorotate synthetase with elastase, has been sub-cloned into the expression vector pCW12 which was then used to transform Escherichia coli S phi 1263 pyrC- lacking dihydroorotase activity. However, induction of this recombinant strain with IPTG produced large amounts of the dihydroorotase domain which were completely inactive. A number of cDNAs were expressed which were longer on the C-terminal side; all cDNAs expressed active dihydroorotase domain down to a minimal extension of 12 amino acids (-Val-Pro-Pro-Gly-Tyr-Gly-Gln-Asp-Val-Arg-Lys-Trp) into the bridge region between the dihydroorotase and aspartate transcarbamylase domains. Part of this dodecapeptide may form an amphipathic helix which in some way constrains the isolated, recombinant dihydroorotase domain to an active conformation. The recombinant hamster dihydroorotase purified from a cell-free extract of E. coli in four steps has a turnover number of 297 mol/min/(mol domain) for the conversion of L-dihydroorotate back to N-carbamyl-L-aspartate with Ks = 8.7 +/- 1.5 microM for L-dihydroorotate, a subunit molecular weight of 39,008 determined from the sequence and 37,900 +/- 400 when subjected to SDS-PAGE, and an isoelectric point of 5.7. Ultracentrifugal analysis of the recombinant domain showed a single species of s20,w = 4.1 S and a single molecular species of M(r) = 76,000 corresponding to a dimer.
Williams
N K
NK
Department of Biochemistry, University of Sydney, NSW, Australia.
Peide
Y
Y
Seymour
K K
KK
Ralston
G B
GB
Christopherson
R I
RI
D000595:Amino Acid Sequence; D000818:Animals; D001221:Aspartate Carbamoyltransferase / Q000235:genetics*; D001483:Base Sequence; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000235:genetics*; D003001:Cloning, Molecular; D006224:Cricetinae; D004080:Dihydroorotase / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics*; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D014162:Transfection
C033626:CAD trifunctional enzyme; D009097:Multienzyme Complexes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D001221:Aspartate Carbamoyltransferase; D004080:Dihydroorotase; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)