Recombinant expression of N‐terminal truncated mutants of the membrane bound mouse, rat and human flavoenzyme dihydroorotate dehydrogenase

Abstract

Mammalian dihydroorotate dehydrogenase, the fourth enzyme of pyrimidine de novo synthesis is an integral protein of the inner mitochondrial membrane that faces the intermembrane space and is functionally connected to the respiratory chain via ubiquinone. Here, we describe the first cloning and analyzing of the complete cDNA of mouse dihydroorotate dehydrogenase. Based on our recent functional expression of the full-length rat and human dihydroorotate dehydrogenase, here we expressed N-terminal-truncated C-terminal-histidine-tagged constructs of the mouse, rat and human enzymes in Escherichia coli. These proteins were devoid of the N-terminal bipartite sequence consisting of the mitochondrial targeting sequence and adjacent hydrophobic domain necessary for import and proper location and fixation of the enzyme in the inner mitochondrial membrane. By employing metal-chelate affinity chromatography under native conditions, the enzymes were purified without detergents to a specific activity of more than 100 mmol´min 21´m g 21 at pH optimum of 8.0±8.1. Flavin analyses by UV-visible spectrometry of the native enzymes gave fairly stoichiometric ratios of 0.6±1.2 mol flavin per mol protein. The kinetic constants of the truncated rat enzyme (K m 11 mm dihydroorotate; K m 7 mm ubiquinone) and human enzyme (K m 10 mm dihydroorotate; K m 14 mm ubiquinone) were very close to those recently reported for the full-size enzymes. The constants for the mouse enzyme, K m 26 mm dihydroorotate and K m 62 mm ubiquinone, were slightly elevated in comparison to those of the other species. The three truncated enzymes were tested for their efficacy with five inhibitors of topical clinical relevance against autoimmune disorders and tumors. Whereas the presence of the N-terminus of dihydroorotate dehydrogenase was essentially irrelevant for the efficacy of the malononitrilamides A77-1726, MNA715 and MNA279 with the rat and human enzyme, the N-termini were found to be important for the efficacy of the dianisidine derivative redoxal. Moreover, the complete N-terminal part of the human enzyme seemed to be of crucial importance for the`slow-binding' features of the cinchoninic acid derivative brequinar, which was suggested to be one of the reasons for the narrow therapeutic window reported from clinical trials on its anti-proliferative and immunosuppressive action.

Malarial Dihydroorotate Dehydrogenase

“…The IC 50 values for inhibition of the malarial DHODH by CCHNP, CCHTFP, and CCHCP are 2200-, 5700-, and 2000-fold higher then the values measured for the recombinant truncated human enzyme under the same assay conditions (Table II). Previously, IC 50 values for these compounds had been reported for the rodent and human enzymes (24,41). The values for the human enzyme are in good agreement with these previous reports.…”

“…Recombinant truncated human DHODH was prepared as described under "Experimental Procedures" for comparative purposes. Steadystate constants were determined for the purified recombinant human enzyme (k cat ϭ 11 Ϯ 0.4 s Ϫ1 and K m app (L-DHO) ϭ 32 Ϯ 4 M) using the DCIP assay; these values are similar to previously reported values for this enzyme (40,41). The IC 50 values for inhibition of the malarial DHODH by CCHNP, CCHTFP, and CCHCP are 2200-, 5700-, and 2000-fold higher then the values measured for the recombinant truncated human enzyme under the same assay conditions (Table II).…”

“…The values for the human enzyme are in good agreement with these previous reports. The IC 50 values for human DHODH are typically 4-to 10-fold higher then those values measured on the rodent enzymes (41).…”

“…Soluble overexpression of human DHODH was achieved by truncating 30 amino acids from the N-terminal membrane anchor (40). The truncation was demonstrated not to affect inhibitor binding affinity, nor enzyme activity (41). The x-ray structure of human DHODH (32) also provides evidence that this truncated protein contains the functional catalytic domain (Fig.…”

“…The PCR product was extracted using a gel purification kit (Qiagen, Valencia, CA) and ligated into a similarly restricted and purified pET22b (Novagen, Madison, WI) protein overexpression vector to generate a construct with a His 6 C-terminal tag. This construct is similar to previously described expression constructs for truncated human DHODH (33,40,41). Transformed BL21(DE3) cells were grown at 37°C in rich LB medium ( Enzymatic Assays for DHODH Activity-Steady-state measurements to determine the pH dependence of enzyme activity were performed by varying the L-DHO concentration (5-500 M) and measuring the reduction of 2,6-dichloroindophenol (DCIP) at 600 nm (⑀ ϭ 18.8 mM Ϫ1 cm Ϫ1 ) as described (38).…”