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 di gestion of dihydroorotate synthetase with elastase, has been sub-clone d into the expression vector pCW12 which was then used to transform Es cherichia coli SO1263 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 extensi on 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 tran scarbamylase domains. Part of this dodecapeptide may form an amphipath ic helix which in some way constrains the isolated, recombinant dihydr oorotase domain to an active conformation. The recombinant hamster dih ydroorotase 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 o f L-dihydroorotate back to N-carbamyl-L-aspartate with K(s) = 8.7 +/- 1.5 muM for L-dihydroorotate, a subunit molecular weight of 39 008 det ermined from the sequence and 37 900 +/- 400 when subjected to SDS-PAG E, and an isoelectric point of 5.7. Ultracentrifugal analysis of the r ecombinant domain showed a single species of s20,w = 4.1 S and a singl e molecular species of M(r) = 76 000 corresponding to a dimer.