{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/84904"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/84904","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Genetic and Biochemical Characterization of the Heteromeric Dihydroorotate Dehydrogenase From Bacillus Subtilis","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.","abstract_html":"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.","abstract_has_math":false,"creators":["Kahler, Andrea Elise"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Biochemistry","degree_department":null,"school":null,"contributors":["Switzer, Robert L."],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2015,"date_issued":"2015-09-25T22:28:26Z","date_published":"2015-09-25T22:28:26Z","updated_at":"2026-07-22T22:26:24Z","subjects":["Biology, Microbiology"],"languages":["eng"],"rights":[],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["(MiAaPQ)AAI9944900"],"render_values":[{"text":"(MiAaPQ)AAI9944900","href":null,"code":true}]}]},"links":{"outbound_url":"http://hdl.handle.net/2142/84904","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Switzer, Robert L."]},{"key":"dc:creator","label":"Author","values":["Kahler, Andrea Elise"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2015-09-25T22:28:26Z","10000-01-01","1999"]},{"key":"dc:type","label":"Dc Type","values":["text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Biochemistry"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["University of Illinois at Urbana-Champaign"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Biology, Microbiology"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://hdl.handle.net/2142/84904","(MiAaPQ)AAI9944900"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["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.","Made available in DSpace on 2015-09-25T22:28:26Z (GMT). No. of bitstreams: 2 license.txt: 4848 bytes, checksum: 96035ab3f5e1c23cc7138a224ce498bd (MD5) 9944900.pdf: 7508179 bytes, checksum: d5c8a7371d85481ce288ae3a11259e43 (MD5) Previous issue date: 1999","Embargo set by: Seth Robbins for item 86185 Lift date: Forever Reason: Restricted to the U of I community idenfinitely during batch ingest of legacy ETDs","Restricted to the U of I community idenfinitely during batch ingest of legacy ETDs","U of I Only","131 p.","Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1999."]},{"key":"dc:title","label":"Title","values":["Genetic and Biochemical Characterization of the Heteromeric Dihydroorotate Dehydrogenase From Bacillus Subtilis"]}]}],"canonical_facts":{"dc:contributor":["Switzer, Robert L."],"dc:creator":["Kahler, Andrea Elise"],"dc:date":["2015-09-25T22:28:26Z","10000-01-01","1999"],"dc:description":["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.","Made available in DSpace on 2015-09-25T22:28:26Z (GMT). No. of bitstreams: 2 license.txt: 4848 bytes, checksum: 96035ab3f5e1c23cc7138a224ce498bd (MD5) 9944900.pdf: 7508179 bytes, checksum: d5c8a7371d85481ce288ae3a11259e43 (MD5) Previous issue date: 1999","Embargo set by: Seth Robbins for item 86185 Lift date: Forever Reason: Restricted to the U of I community idenfinitely during batch ingest of legacy ETDs","Restricted to the U of I community idenfinitely during batch ingest of legacy ETDs","U of I Only","131 p.","Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1999."],"dc:identifier":["http://hdl.handle.net/2142/84904","(MiAaPQ)AAI9944900"],"dc:language":["eng"],"dc:subject":["Biology, Microbiology"],"dc:title":["Genetic and Biochemical Characterization of the Heteromeric Dihydroorotate Dehydrogenase From Bacillus Subtilis"],"dc:type":["text"],"thesis:degree_discipline":["Biochemistry"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Ph.D."],"thesis:institution_name":["University of Illinois at Urbana-Champaign"]},"updated_at":"2026-07-22T22:26:24Z"}