Genetic and Biochemical Characterization of the Heteromeric Dihydroorotate Dehydrogenase From Bacillus Subtilis

Co-expression and purification of PyrDI (Mr = 33,094) and PyrDII (Mr = 28,099) in E. coli demonstrated that the two proteins formed a heteromeric DHOD holoenzyme, an iron-sulfur flavoprotein which was determined by gel filtration to be a tetramer containing 2 mol PyrDI and 2 mol PyrDII. The two subunits were also overexpressed individually and purified. Overexpressed PyrDII formed inclusion bodies and could be purified by refolding and reconstitution with cofactors. Purified PyrDI was a flavoprotein, and refolded PyrDII bound 1 mol FAD and 1 mol [2Fe-2S] per mol subunit. 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 greater 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 regenerated by mixing the individually purified subunits, which bound with an estimated Kd value of 19 +/- 9 nM. Activity regenerated from the subunit mixtures showed a clear dependence on FAD reconstitution of PyrDII but not on its reconstitution with iron-sulfur 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 predicted cysteine ligands to the [2Fe-2S] cluster failed to complement the pyr bradytrophy of a B. subtilis Delta pyrDII strain, indicating a requirement for the iron-sulfur cluster in PyrDII for normal function in vivo.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biochemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2015

Author dc:creator

• Kahler, Andrea Elise

Contributors dc:contributor

• Switzer, Robert L.

• Biology, Microbiology

Language dc:language

eng

Identifier

(MiAaPQ)AAI9944900

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/84904