{"id":{"repo_id":"vt","oai_identifier":"oai:vtechworks.lib.vt.edu:10919/76837"},"canonical_url":"https://search.dev.ndltd.org/etd/vt/oai:vtechworks.lib.vt.edu:10919/76837","repository":{"repo_id":"vt","name":"Virginia Tech","base_url":"https://vtechworks.lib.vt.edu/oai/request"},"display":{"title":"Structural and Mutational Analyses of Aspergillus fumigatus SidA: A Flavin-Dependent N-hydroxylating Enzyme","abstract":"SidA from Aspergillus fumigatus is an N-hydroxylating monooxygenase that catalyzes the committed step in siderophore biosynthesis. This gene is essential for virulence making it an excellent drug target. In order to design an inhibitor against SidA a greater understanding of the mechanism and structure is needed. We have determined the crystal structure of SidA in complex with NADP+, Ornithine, and FAD at 1.9 ? resolution. The crystal structure has provided insight into substrate and coenzyme selectivity as well as residues essential for catalysis. In particular, we have chosen to study the interactions of Arg 279, shown to interact with the 2'phosphate of the adenine moiety of NADP+ as well as the adenine ring itself. The mutation of this residue to alanine makes the enzyme have little to no selectivity between coenzymes NADPH and NADH which supports the importance of the ionic interaction between Arg279 and the 2'phosphate. Additionally, the mutant enzyme is significantly more uncoupled than WT enzyme with NADPH. We see that the interactions of the guanadinyl group of Arg279 and the adenine ring are also important because KM and Kd values for the mutant enzyme are shifted well above those of wild type with coenzyme NADH. The data is further supported by studies on the reductive and oxidative half reactions. We have also explored the allosteric effect of L-arginine. We provide evidence that an enzyme/coenzyme/L-arginine complex is formed which improves coupling, oxygen reactivity, and reduction in SidA; however more work is needed to fully understand the role of L-arginine as an allosteric effector.","abstract_html":"SidA from Aspergillus fumigatus is an N-hydroxylating monooxygenase that catalyzes the committed step in siderophore biosynthesis. This gene is essential for virulence making it an excellent drug target. In order to design an inhibitor against SidA a greater understanding of the mechanism and structure is needed. We have determined the crystal structure of SidA in complex with NADP+, Ornithine, and FAD at 1.9 ? resolution. The crystal structure has provided insight into substrate and coenzyme selectivity as well as residues essential for catalysis. In particular, we have chosen to study the interactions of Arg 279, shown to interact with the 2'phosphate of the adenine moiety of NADP+ as well as the adenine ring itself. The mutation of this residue to alanine makes the enzyme have little to no selectivity between coenzymes NADPH and NADH which supports the importance of the ionic interaction between Arg279 and the 2'phosphate. Additionally, the mutant enzyme is significantly more uncoupled than WT enzyme with NADPH. We see that the interactions of the guanadinyl group of Arg279 and the adenine ring are also important because KM and Kd values for the mutant enzyme are shifted well above those of wild type with coenzyme NADH. The data is further supported by studies on the reductive and oxidative half reactions. We have also explored the allosteric effect of L-arginine. We provide evidence that an enzyme/coenzyme/L-arginine complex is formed which improves coupling, oxygen reactivity, and reduction in SidA; however more work is needed to fully understand the role of L-arginine as an allosteric effector.","abstract_has_math":false,"creators":["Fedkenheuer, Michael Gerald"],"institution":"Virginia Tech","degree_name":"Master of Science in Life Sciences","degree_level":"masters","degree_discipline":"Biochemistry","degree_department":"Biochemistry","school":null,"contributors":[],"advisors":[],"committee_chairs":["Sobrado, Pablo"],"committee_members":["Finkielstein, Carla V.","Xu, Bu"],"year":2012,"date_issued":"2012-07-27","date_published":"2012-07-27","updated_at":"2026-07-22T22:19:42Z","subjects":["N-hydroxylating Monooxygenases","Af SidA","hydroxylation","flavin","siderophore"],"languages":["en_US"],"rights":["In Copyright"],"rights_urls":["http://rightsstatements.org/vocab/InC/1.0/"],"identifier_entries":[{"key":"dc:identifier.other","label":"Dc Identifier Other","values":["etd-08082012-192654"],"render_values":[{"text":"etd-08082012-192654","href":null,"code":true}]}]},"links":{"outbound_url":"http://hdl.handle.net/10919/76837","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.committeechair","label":"Committee Chair","values":["Sobrado, Pablo"]},{"key":"dc:contributor.committeemember","label":"Committee Member","values":["Finkielstein, Carla V.","Xu, Bu"]},{"key":"dc:contributor.department","label":"Department","values":["Biochemistry"]},{"key":"dc:creator","label":"Author","values":["Fedkenheuer, Michael Gerald"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2017-04-04T19:49:37Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2017-04-04T19:49:37Z","2016-09-30"]},{"key":"dc:date.issued","label":"Date","values":["2012-07-27"]},{"key":"dc:publisher","label":"Institution","values":["Virginia Tech"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.dcmitype","label":"Dc Type Dcmitype","values":["Text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Biochemistry"]},{"key":"thesis:degree_level","label":"Degree Level","values":["masters"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Master of Science in Life Sciences"]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["Virginia Polytechnic Institute and State University"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["N-hydroxylating Monooxygenases","Af SidA","hydroxylation","flavin","siderophore"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language.iso","label":"Language (ISO)","values":["en_US"]},{"key":"dc:rights","label":"Dc Rights","values":["In Copyright"]},{"key":"dc:rights.uri","label":"Rights URI","values":["http://rightsstatements.org/vocab/InC/1.0/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.other","label":"Dc Identifier Other","values":["etd-08082012-192654"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["http://hdl.handle.net/10919/76837"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["SidA from Aspergillus fumigatus is an N-hydroxylating monooxygenase that catalyzes the committed step in siderophore biosynthesis. This gene is essential for virulence making it an excellent drug target. In order to design an inhibitor against SidA a greater understanding of the mechanism and structure is needed. We have determined the crystal structure of SidA in complex with NADP+, Ornithine, and FAD at 1.9 ? resolution. The crystal structure has provided insight into substrate and coenzyme selectivity as well as residues essential for catalysis. In particular, we have chosen to study the interactions of Arg 279, shown to interact with the 2'phosphate of the adenine moiety of NADP+ as well as the adenine ring itself. The mutation of this residue to alanine makes the enzyme have little to no selectivity between coenzymes NADPH and NADH which supports the importance of the ionic interaction between Arg279 and the 2'phosphate. Additionally, the mutant enzyme is significantly more uncoupled than WT enzyme with NADPH. We see that the interactions of the guanadinyl group of Arg279 and the adenine ring are also important because KM and Kd values for the mutant enzyme are shifted well above those of wild type with coenzyme NADH. The data is further supported by studies on the reductive and oxidative half reactions. We have also explored the allosteric effect of L-arginine. We provide evidence that an enzyme/coenzyme/L-arginine complex is formed which improves coupling, oxygen reactivity, and reduction in SidA; however more work is needed to fully understand the role of L-arginine as an allosteric effector."]},{"key":"dc:description.degree","label":"Dc Description Degree","values":["Master of Science in Life Sciences"]},{"key":"dc:title","label":"Title","values":["Structural and Mutational Analyses of Aspergillus fumigatus SidA: A Flavin-Dependent N-hydroxylating Enzyme"]}]}],"canonical_facts":{"dc:contributor.committeechair":["Sobrado, Pablo"],"dc:contributor.committeemember":["Finkielstein, Carla V.","Xu, Bu"],"dc:contributor.department":["Biochemistry"],"dc:creator":["Fedkenheuer, Michael Gerald"],"dc:date.accessioned":["2017-04-04T19:49:37Z"],"dc:date.available":["2017-04-04T19:49:37Z","2016-09-30"],"dc:date.issued":["2012-07-27"],"dc:description.abstract":["SidA from Aspergillus fumigatus is an N-hydroxylating monooxygenase that catalyzes the committed step in siderophore biosynthesis. This gene is essential for virulence making it an excellent drug target. In order to design an inhibitor against SidA a greater understanding of the mechanism and structure is needed. We have determined the crystal structure of SidA in complex with NADP+, Ornithine, and FAD at 1.9 ? resolution. The crystal structure has provided insight into substrate and coenzyme selectivity as well as residues essential for catalysis. In particular, we have chosen to study the interactions of Arg 279, shown to interact with the 2'phosphate of the adenine moiety of NADP+ as well as the adenine ring itself. The mutation of this residue to alanine makes the enzyme have little to no selectivity between coenzymes NADPH and NADH which supports the importance of the ionic interaction between Arg279 and the 2'phosphate. Additionally, the mutant enzyme is significantly more uncoupled than WT enzyme with NADPH. We see that the interactions of the guanadinyl group of Arg279 and the adenine ring are also important because KM and Kd values for the mutant enzyme are shifted well above those of wild type with coenzyme NADH. The data is further supported by studies on the reductive and oxidative half reactions. We have also explored the allosteric effect of L-arginine. We provide evidence that an enzyme/coenzyme/L-arginine complex is formed which improves coupling, oxygen reactivity, and reduction in SidA; however more work is needed to fully understand the role of L-arginine as an allosteric effector."],"dc:description.degree":["Master of Science in Life Sciences"],"dc:identifier.other":["etd-08082012-192654"],"dc:identifier.uri":["http://hdl.handle.net/10919/76837"],"dc:language.iso":["en_US"],"dc:publisher":["Virginia Tech"],"dc:rights":["In Copyright"],"dc:rights.uri":["http://rightsstatements.org/vocab/InC/1.0/"],"dc:subject":["N-hydroxylating Monooxygenases","Af SidA","hydroxylation","flavin","siderophore"],"dc:title":["Structural and Mutational Analyses of Aspergillus fumigatus SidA: A Flavin-Dependent N-hydroxylating Enzyme"],"dc:type":["Thesis"],"dc:type.dcmitype":["Text"],"thesis:degree_discipline":["Biochemistry"],"thesis:degree_level":["masters"],"thesis:degree_name":["Master of Science in Life Sciences"],"thesis:institution_name":["Virginia Polytechnic Institute and State University"]},"updated_at":"2026-07-22T22:19:42Z"}