{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/72334"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/72334","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Evolution of (Beta/alpha)8 Barrels: Refinement of Enzymatic Efficiency for a New Ortho-Succinylbenzoate Synthase (Osbs) From a Promiscuous Progenitor","abstract":"This work allowed a step-wise pathway of changing base pairs to be elucidated that illustrates that a minimal amount of mutations (four from wild-type to I19F/R24W/D297G) are necessary to develop a new function. Additionally, the "new" function of OSBS activity was achieved with changes to substrate specificity, the substitutions allowed the catalytic barrel to remain "hard-wired" to perform acid/base chemistry as both the AEE and OSBS reactions utilized the same catalytic lysines.","abstract_html":"This work allowed a step-wise pathway of changing base pairs to be elucidated that illustrates that a minimal amount of mutations (four from wild-type to I19F/R24W/D297G) are necessary to develop a new function. Additionally, the "new" function of OSBS activity was achieved with changes to substrate specificity, the substitutions allowed the catalytic barrel to remain "hard-wired" to perform acid/base chemistry as both the AEE and OSBS reactions utilized the same catalytic lysines.","abstract_has_math":false,"creators":["Vick, Jacob E."],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Biochemistry","degree_department":null,"school":null,"contributors":["Gerlt, John A."],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2014,"date_issued":"2014-12-17T21:58:29Z","date_published":"2014-12-17T21:58:29Z","updated_at":"2026-07-22T22:26:06Z","subjects":["Chemistry, Biochemistry"],"languages":[],"rights":[],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["(UMI)AAI3314925"],"render_values":[{"text":"(UMI)AAI3314925","href":null,"code":true}]}]},"links":{"outbound_url":"http://hdl.handle.net/2142/72334","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":["Vick, Jacob E."]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2014-12-17T21:58:29Z","10000-01-01","2008"]},{"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":["Chemistry, Biochemistry"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://hdl.handle.net/2142/72334","(UMI)AAI3314925"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["This work allowed a step-wise pathway of changing base pairs to be elucidated that illustrates that a minimal amount of mutations (four from wild-type to I19F/R24W/D297G) are necessary to develop a new function. Additionally, the "new" function of OSBS activity was achieved with changes to substrate specificity, the substitutions allowed the catalytic barrel to remain "hard-wired" to perform acid/base chemistry as both the AEE and OSBS reactions utilized the same catalytic lysines.","Made available in DSpace on 2014-12-17T21:58:29Z (GMT). No. of bitstreams: 1 3314925.pdf: 2757415 bytes, checksum: 8f38c46152aa3cfde8dd35f1b4da4aeb (MD5) Previous issue date: 2008","To explore how an enzyme can gain a new function, the L-Ala-D/L-Glu epimerase (AEE) from Escherichia coli was chosen as the starting point for the development of a "new" o-succinylbenzoate synthase (OSBS). AEE and OSBS are members of the enolase superfamily that have a conserved enzyme structure including a (beta/alpha)8-barrel "catalytic domain" and a N-terminal "capping domain" for substrate specificity. Members of the enolase superfamily all catalyze reactions that share the abstraction from a carbon alpha to a carboxylic acid to produce a divalent metal stabilized enolate anion intermediate.","Using structural alignments, the D297G mutation was found to allow a low level of OSBS activity while reducing the efficiency of the AEE reaction [Schmidt, D. M. Z. et al. (2003) Biochemistry 42, 8387--8393]. The D297G mutant allowed an 8 x 107 improvement in catalytic rate by allowing access of the 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid (SHCHC) substrate by removing unfavorable steric and charge interactions. Anaerobic selection of an error-prone library identified the I19F mutant that allowed for an additional 12-fold increase in catalytic efficiency. The I19F mutant is located in the 20s loop of the capping domain that sequesters the active site from solvent [Vick, J. E., Schmidt, D. M. Z., and Gerlt, J. A. (2005) Biochemistry 44, 11722--11729]. Additional anaerobic selection identified the R24C and L277W mutants that each increased the OSBS efficiency of the I19F/D297G mutant [Vick, J. E. and Gerlt, J. A. (2007) Biochemistry 46, 14589--14598]. These mutants appear to function by altering the interaction between I19F and the substrate/product. Using site-specific randomization, R24W, a single base pair change from R24C, was identified as the greatest improvement of catalytic efficiency to date.","Embargo set by: Seth Robbins for item 72502 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","181 p.","Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2008."]},{"key":"dc:title","label":"Title","values":["Evolution of (Beta/alpha)8 Barrels: Refinement of Enzymatic Efficiency for a New Ortho-Succinylbenzoate Synthase (Osbs) From a Promiscuous Progenitor"]}]}],"canonical_facts":{"dc:contributor":["Gerlt, John A."],"dc:creator":["Vick, Jacob E."],"dc:date":["2014-12-17T21:58:29Z","10000-01-01","2008"],"dc:description":["This work allowed a step-wise pathway of changing base pairs to be elucidated that illustrates that a minimal amount of mutations (four from wild-type to I19F/R24W/D297G) are necessary to develop a new function. Additionally, the "new" function of OSBS activity was achieved with changes to substrate specificity, the substitutions allowed the catalytic barrel to remain "hard-wired" to perform acid/base chemistry as both the AEE and OSBS reactions utilized the same catalytic lysines.","Made available in DSpace on 2014-12-17T21:58:29Z (GMT). No. of bitstreams: 1 3314925.pdf: 2757415 bytes, checksum: 8f38c46152aa3cfde8dd35f1b4da4aeb (MD5) Previous issue date: 2008","To explore how an enzyme can gain a new function, the L-Ala-D/L-Glu epimerase (AEE) from Escherichia coli was chosen as the starting point for the development of a "new" o-succinylbenzoate synthase (OSBS). AEE and OSBS are members of the enolase superfamily that have a conserved enzyme structure including a (beta/alpha)8-barrel "catalytic domain" and a N-terminal "capping domain" for substrate specificity. Members of the enolase superfamily all catalyze reactions that share the abstraction from a carbon alpha to a carboxylic acid to produce a divalent metal stabilized enolate anion intermediate.","Using structural alignments, the D297G mutation was found to allow a low level of OSBS activity while reducing the efficiency of the AEE reaction [Schmidt, D. M. Z. et al. (2003) Biochemistry 42, 8387--8393]. The D297G mutant allowed an 8 x 107 improvement in catalytic rate by allowing access of the 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid (SHCHC) substrate by removing unfavorable steric and charge interactions. Anaerobic selection of an error-prone library identified the I19F mutant that allowed for an additional 12-fold increase in catalytic efficiency. The I19F mutant is located in the 20s loop of the capping domain that sequesters the active site from solvent [Vick, J. E., Schmidt, D. M. Z., and Gerlt, J. A. (2005) Biochemistry 44, 11722--11729]. Additional anaerobic selection identified the R24C and L277W mutants that each increased the OSBS efficiency of the I19F/D297G mutant [Vick, J. E. and Gerlt, J. A. (2007) Biochemistry 46, 14589--14598]. These mutants appear to function by altering the interaction between I19F and the substrate/product. Using site-specific randomization, R24W, a single base pair change from R24C, was identified as the greatest improvement of catalytic efficiency to date.","Embargo set by: Seth Robbins for item 72502 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","181 p.","Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2008."],"dc:identifier":["http://hdl.handle.net/2142/72334","(UMI)AAI3314925"],"dc:subject":["Chemistry, Biochemistry"],"dc:title":["Evolution of (Beta/alpha)8 Barrels: Refinement of Enzymatic Efficiency for a New Ortho-Succinylbenzoate Synthase (Osbs) From a Promiscuous Progenitor"],"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":"2026-07-22T22:26:06Z"}