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.

Location of the dihydroorotase domain within trifunctional hamster dihydroorotate synthetase.

Mammalian dihydroorotase (DHOase, EC 3.5.2.3) is part of a trifunctional protein, dihydroorotate synthetase which catalyzes the first three reactions of de novo pyrimidine biosynthesis. We have subcloned a portion of the cDNA from the plasmid pCAD142 and obtained a nucleotide sequence which extends 2.1 kb in the 5' direction from the sequence encoding the aspartate transcarbamoylase (ATCase) domain at the 3'-end of the cDNA. The DHOase and ATCase domains have been purified from an elastase digest of the trifunctional protein and subjected to amino acid (aa) sequencing from their N termini. The sequence of the N-terminal 24 aa of the DHOase domain has been obtained and aligned with the cDNA sequence. The C-terminal residues of the DHOase domain have been identified as Leu followed by Val which, when taken with partial sequences of the CNBr fragments of this domain, defines the coding sequence of the active, globular DHOase domain released by proteolysis. Prediction of protein secondary structure from the deduced aa sequence showed that the DHOase domain (Mr 37,751) is separated from the C-terminal ATCase domain (Mr 34,323) by a bridging sequence (Mr 12,532) consisting of multiple beta-turns.

Purification of hamster dihydroorotate synthetase using Procion blue-Sepharose.

Dihydroorotate (DHO) synthetase is a trifunctional protein that catalyzes the first three reactions of de novo pyrimidine biosynthesis. A single-step procedure for purification of DHO synthetase from mutant hamster cells that overproduce this protein has been developed. The synthetase is adsorbed from a postmitochondrial supernatant to a column of Procion blue-Sepharose 4B and, after the column is washed, the synthetase is eluted as a single peak with 0.4 M KCl. Pooled fractions from the trailing side of this peak yield DHO synthetase with a specific activity for aspartate transcarbamylase of 14 mumol/min/mg protein, representing a purification factor of 8.5-fold and a recovery of 28% from the postmitochondrial supernatant. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the DHO synthetase was of high purity. A further 34% of the DHO synthetase from the leading side of the eluted peak contained a minor proportion of a proteolytic fragment. Similar results were obtained with an established four-step purification procedure.