{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/84141"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/84141","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Characterization of Ortho-Succinylbenzoate Synthase (Osbs): A Study of Mechanism, Proficiency and Evolutionary Diversity","abstract":"\"ortho-Succinylbenzoate synthase (OSBS), an enzyme of the enolase superfamily, catalyzes a dehydration reaction in the menaquinone biosynthetic pathway. Menaquinone is an electron acceptor that is utilized in bacteria, archaebacteria and red green algae. OSBSs from over 70 organisms have been identified and their pairwise sequence homology is often as low as 14%. The sequence divergence has allowed us to explore enzymes with different sequences that catalyze the same reaction, thus providing insight into evolutionary paths used within the enolase superfamily. To understand the sequence diversity of OSBSs, the enzymatic contribution to catalysis was determined. The enzymatic proficiency was 102--106 fold lower than for other enzymes that perform mechanistically similar reactions. The relatively low enzymatic proficiency could provide less stringent selective pressure on the enzyme, allowing for divergence in the sequences of OSBSs. The OSBS from E. coli was explored through kinetic isotope experiments and mutational analysis to determine the overall mechanism of the reaction to be a stepwise syn elimination reaction. Kinetic isotope studies showed a large primary isotope effect suggesting a mechanism dominated by alpha-proton abstraction. Site-directed mutation of active site Lys residues confirmed assignment of the catalytic residues. Additionally, the K133R mutant was co-crystallized with the substrate allowing the stereoconfiguration to be determined. The OSBS from Amycolaptosis is a promiscuous enzyme that can also function as an \"\"N-acylamino acid racemase\"\" (NAAAR). The adventitious racemization reaction catalyzed by this OSBS was characterized in addition to the native reaction. The discovery of a naturally promiscuous enzyme provides support to the theory that enzyme evolution proceeds through intermediates that maintain their native function but that have evolved to catalyze additional reactions. Exploration of these two enzymes, in addition to other OSBS orthologues, has lead to the expansion of structural and mechanistic understanding of the divergent evolution in the enolase superfamily.\"","abstract_html":""ortho-Succinylbenzoate synthase (OSBS), an enzyme of the enolase superfamily, catalyzes a dehydration reaction in the menaquinone biosynthetic pathway. Menaquinone is an electron acceptor that is utilized in bacteria, archaebacteria and red green algae. OSBSs from over 70 organisms have been identified and their pairwise sequence homology is often as low as 14%. The sequence divergence has allowed us to explore enzymes with different sequences that catalyze the same reaction, thus providing insight into evolutionary paths used within the enolase superfamily. To understand the sequence diversity of OSBSs, the enzymatic contribution to catalysis was determined. The enzymatic proficiency was 102--106 fold lower than for other enzymes that perform mechanistically similar reactions. The relatively low enzymatic proficiency could provide less stringent selective pressure on the enzyme, allowing for divergence in the sequences of OSBSs. The OSBS from E. coli was explored through kinetic isotope experiments and mutational analysis to determine the overall mechanism of the reaction to be a stepwise syn elimination reaction. Kinetic isotope studies showed a large primary isotope effect suggesting a mechanism dominated by alpha-proton abstraction. Site-directed mutation of active site Lys residues confirmed assignment of the catalytic residues. Additionally, the K133R mutant was co-crystallized with the substrate allowing the stereoconfiguration to be determined. The OSBS from Amycolaptosis is a promiscuous enzyme that can also function as an ""N-acylamino acid racemase"" (NAAAR). The adventitious racemization reaction catalyzed by this OSBS was characterized in addition to the native reaction. The discovery of a naturally promiscuous enzyme provides support to the theory that enzyme evolution proceeds through intermediates that maintain their native function but that have evolved to catalyze additional reactions. Exploration of these two enzymes, in addition to other OSBS orthologues, has lead to the expansion of structural and mechanistic understanding of the divergent evolution in the enolase superfamily."","abstract_has_math":false,"creators":["Taylor Ringia, Erika Anne"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Chemistry","degree_department":null,"school":null,"contributors":["Gerlt, John A."],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2015,"date_issued":"2015-09-25T22:13:08Z","date_published":"2015-09-25T22:13:08Z","updated_at":"2026-07-22T22:26:22Z","subjects":["Biophysics, General"],"languages":["eng"],"rights":[],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["(MiAaPQ)AAI3131036"],"render_values":[{"text":"(MiAaPQ)AAI3131036","href":null,"code":true}]}]},"links":{"outbound_url":"http://hdl.handle.net/2142/84141","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Gerlt, John A."]},{"key":"dc:creator","label":"Author","values":["Taylor Ringia, Erika Anne"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2015-09-25T22:13:08Z","10000-01-01","2004"]},{"key":"dc:type","label":"Dc Type","values":["text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Chemistry"]},{"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":["Biophysics, General"]}]},{"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/2142/84141","(MiAaPQ)AAI3131036"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["\"ortho-Succinylbenzoate synthase (OSBS), an enzyme of the enolase superfamily, catalyzes a dehydration reaction in the menaquinone biosynthetic pathway. Menaquinone is an electron acceptor that is utilized in bacteria, archaebacteria and red green algae. OSBSs from over 70 organisms have been identified and their pairwise sequence homology is often as low as 14%. The sequence divergence has allowed us to explore enzymes with different sequences that catalyze the same reaction, thus providing insight into evolutionary paths used within the enolase superfamily. To understand the sequence diversity of OSBSs, the enzymatic contribution to catalysis was determined. The enzymatic proficiency was 102--106 fold lower than for other enzymes that perform mechanistically similar reactions. The relatively low enzymatic proficiency could provide less stringent selective pressure on the enzyme, allowing for divergence in the sequences of OSBSs. The OSBS from E. coli was explored through kinetic isotope experiments and mutational analysis to determine the overall mechanism of the reaction to be a stepwise syn elimination reaction. Kinetic isotope studies showed a large primary isotope effect suggesting a mechanism dominated by alpha-proton abstraction. Site-directed mutation of active site Lys residues confirmed assignment of the catalytic residues. Additionally, the K133R mutant was co-crystallized with the substrate allowing the stereoconfiguration to be determined. The OSBS from Amycolaptosis is a promiscuous enzyme that can also function as an \"\"N-acylamino acid racemase\"\" (NAAAR). The adventitious racemization reaction catalyzed by this OSBS was characterized in addition to the native reaction. The discovery of a naturally promiscuous enzyme provides support to the theory that enzyme evolution proceeds through intermediates that maintain their native function but that have evolved to catalyze additional reactions. Exploration of these two enzymes, in addition to other OSBS orthologues, has lead to the expansion of structural and mechanistic understanding of the divergent evolution in the enolase superfamily.\"","Made available in DSpace on 2015-09-25T22:13:08Z (GMT). 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Menaquinone is an electron acceptor that is utilized in bacteria, archaebacteria and red green algae. OSBSs from over 70 organisms have been identified and their pairwise sequence homology is often as low as 14%. The sequence divergence has allowed us to explore enzymes with different sequences that catalyze the same reaction, thus providing insight into evolutionary paths used within the enolase superfamily. To understand the sequence diversity of OSBSs, the enzymatic contribution to catalysis was determined. The enzymatic proficiency was 102--106 fold lower than for other enzymes that perform mechanistically similar reactions. The relatively low enzymatic proficiency could provide less stringent selective pressure on the enzyme, allowing for divergence in the sequences of OSBSs. The OSBS from E. coli was explored through kinetic isotope experiments and mutational analysis to determine the overall mechanism of the reaction to be a stepwise syn elimination reaction. Kinetic isotope studies showed a large primary isotope effect suggesting a mechanism dominated by alpha-proton abstraction. Site-directed mutation of active site Lys residues confirmed assignment of the catalytic residues. Additionally, the K133R mutant was co-crystallized with the substrate allowing the stereoconfiguration to be determined. The OSBS from Amycolaptosis is a promiscuous enzyme that can also function as an \"\"N-acylamino acid racemase\"\" (NAAAR). The adventitious racemization reaction catalyzed by this OSBS was characterized in addition to the native reaction. The discovery of a naturally promiscuous enzyme provides support to the theory that enzyme evolution proceeds through intermediates that maintain their native function but that have evolved to catalyze additional reactions. Exploration of these two enzymes, in addition to other OSBS orthologues, has lead to the expansion of structural and mechanistic understanding of the divergent evolution in the enolase superfamily.\"","Made available in DSpace on 2015-09-25T22:13:08Z (GMT). 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