Details

Autor(en) / Beteiligte

• Kahler, Andrea E
• Nielsen, Finn S
• Switzer, Robert L

Titel

Biochemical Characterization of the Heteromeric Bacillus subtilis Dihydroorotate Dehydrogenase and Its Isolated Subunits

Ist Teil von

• Archives of biochemistry and biophysics, 1999-11, Vol.371 (2), p.191-201

Ort / Verlag

United States: Elsevier Inc

Erscheinungsjahr

1999

Quelle

Elsevier Journal Backfiles on ScienceDirect (DFG Nationallizenzen)

Beschreibungen/Notizen

• Bacillus subtilis dihydroorotate dehydrogenase (DHOD) consists of two subunits, PyrDI (Mr = 33,094) and PyrDII (Mr = 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 (PyrDI2PyrDII2). 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 Km value for dihydroorotate, an approximately 50-fold smaller Ki 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 Kd 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 ΔpyrDII strain, indicating a requirement for the FeS cluster in PyrDII for normal function in vivo.