Replacing the ({beta}{alpha})-unit 8 of E.coli TIM with its chicken homologue leads to a stable and active hybrid enzyme |
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Authors: | Mainfroid Veronique; Goraj Karine; Rentier-Delrue Francoise; Houbrechts Annick; Noble Martin EM; Borchert Torben V; Wierenga Rik K; Martial Joseph A |
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Affiliation: | Laboratoire de Biologic Molèculaire et de Gènie Gènètique, Universitè de Liège, Institut de Chimie B6, B-4000 Sart Tilman, Belgium
1EMBL, Meyerhofstrasse 1 D6900 Heidelberg, Germany |
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Abstract: | In order to investigate how structural modifications interferewith protein stability, we modified a (ß )-unit inE.coli triosephosphate isomerase (TIM), a typical (ß )-barrelprotein, assuming that the pseudosymmetrical ß-barrelcan be divided into eight successive loop/ß-strand/loop/ -helixmotifs. We replaced the eighth (ß )-unit of E.coliTIM with the corresponding chicken (ß )-unit. The substitution,involving the replacement of 10 of the 23 residues of this (ß )-unit, was evaluated first by modelling, then experimentally.Modelling by bomology suggests how the amino add replacementsmight be accommodated in the hybrid E.coli/chicken TIM (ETCM8CHI).Both natural and hybrid recombinant TIMs, overproduced in E.coli,were purified to homogeneity and characterized as to their stabilityand kinetics. Our kinetic studies show that the modificationperformed here leads to an active enzyme. The stability studiesindicate that the stability of ETIM8CHI is comparable to thatof the wild type TIM. |
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Keywords: | (ß )-barrel/" target="_blank">gif" ALT="{alpha}" BORDER="0">)-barrel/ catalytic activity/ protein engineering/ protein folding/ triosephosphate isomerase |
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