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

Bacillus subtilis dihydroorotate dehydrogenase (DHOD) consists of two subun its, PyrDI (M-r = 33,094) and PyrDII (M-r = 28,099). The two subunits were over-expressed jointly and individually and purified. PyrDI was an FMN-cont aining flavoprotein with an apparent native molecular mass of 85,000. Overe xpressed PyrDII formed inclusion bodies and was purified by refolding and r econstitution. Refolded PyrDII bound 1 mol FAD and 1 mol [2Fe-2S] per mol P yrDII. Coexpression and purification of PyrDI and PyrDII yielded a native h oloenzyme complex with an apparent native molecular mass of 114,000 that in dicated a heterotetramer (PyrDI(2)PyrDII(2)). The holoenzyme possessed di-h ydroorotate: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-fol d smaller K-m value for dihydroorotate, an approximately 50-fold smaller K- i value for orotate, and approximately 500-fold greater catalytic efficienc y. Dihydroorotate:NAD(+) oxidoreductase activity could be recovered by mixi ng the purified subunits. Recovered activity showed a clear dependence on F AD reconstitution of PyrDII but not on reconstitution with FeS clusters. Py rDII had a strong preference for FAD over FMN and bound it with an estimate d K-d value of 4.9 +/- 0.8 nM. pyrDII mutants containing alanine substituti ons of the cysteine ligands to the [2Fe-2S] cluster failed to complement th e pyr bradytrophy of a Delta pyrDII strain, indicating a requirement for th e FeS cluster in PyrDII for normal function in vivo. (C) 1999 Academic Pres s.