Genetic and Biochemical Characterization of the Heteromeric Dihydroorotate Dehydrogenase From Bacillus Subtilis
Kahler, Andrea Elise
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https://hdl.handle.net/2142/84904
Description
Title
Genetic and Biochemical Characterization of the Heteromeric Dihydroorotate Dehydrogenase From Bacillus Subtilis
Author(s)
Kahler, Andrea Elise
Issue Date
1999
Doctoral Committee Chair(s)
Switzer, Robert L.
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Microbiology
Language
eng
Abstract
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.
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