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oai:www.ideals.illinois.edu:2142/72334 2023-07-11 col_2142_5131 col_2142_14795 com_2142_5130 com_2142_14794 com_2142_14793 com_2142_8903

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 Gerlt, John A. Vick, Jacob E. 2014-12-17T21:58:29Z 2014-12-17T21:58:29Z 10000-01-01 2008 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. http://hdl.handle.net/2142/72334 (UMI)AAI3314925 Chemistry, Biochemistry Evolution of (Beta/alpha)8 Barrels: Refinement of Enzymatic Efficiency for a New Ortho-Succinylbenzoate Synthase (Osbs) From a Promiscuous Progenitor text Biochemistry Biochemistry University of Illinois at Urbana-Champaign Dissertation Ph.D.