Biochemical characterization of the heteromeric Bacillus subtilis dihydroorotate dehydrogenase and its isolated...

Bacillus subtilis dihydroorotate dehydrogenase (DHOD) consists of two subunits, PyrDI (M(r) = 33,094) and PyrDII (M(r) = 28,099). The two subunits were overexpressed jointly and individually and purified. PyrDI was an FMN-containing flavoprotein with an apparent native molecular mass of 85,000. Overexpressed PyrDII formed inclusion bodies and was purified by refolding and reconstitution. Refolded PyrDII bound 1 mol FAD and 1 mol [2Fe-2S] per mol PyrDII. Coexpression and purification of PyrDI and PyrDII yielded a native holoenzyme complex with an apparent native molecular mass of 114,000 that indicated a heterotetramer (PyrDI(2)PyrDII(2)). The holoenzyme possessed dihydroorotate:NAD(+) oxidoreductase activity and could also reduce menadione and artificial dyes. Purified PyrDI also possessed DHOD activity but could not reduce NAD(+). Compared to PyrDI, the holoenzyme had a more than 20-fold smaller K(m) value for dihydroorotate, an approximately 50-fold smaller K(i) value for orotate, and approximately 500-fold greater catalytic efficiency. Dihydroorotate:NAD(+) oxidoreductase activity could be recovered by mixing the purified subunits. Recovered activity showed a clear dependence on FAD reconstitution of PyrDII but not on reconstitution with FeS clusters. PyrDII had a strong preference for FAD over FMN and bound it with an estimated K(d) value of 4.9 +/- 0.8 nM. pyrDII mutants containing alanine substitutions of the cysteine ligands to the [2Fe-2S] cluster failed to complement the pyr bradytrophy of a DeltapyrDII strain, indicating a requirement for the FeS cluster in PyrDII for normal function in vivo.