Dépôt numérique

Structure-based recombination of beta-lactamases: functional, structural and dynamic investigation of artificially-evolved enzymes

Gobeil, Sophie; Park, Jaeok; Clouthier, Christopher M.; Doucet, Nicolas ORCID logoORCID: https://orcid.org/0000-0002-1952-9380; Berghuis, Albert M. et Pelletier, Joelle N. (2012). Structure-based recombination of beta-lactamases: functional, structural and dynamic investigation of artificially-evolved enzymes In: 26th Annual Symposium of the Protein-Society, August 5–8, 2012, San Diego, California,.

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TEM-1 and PSE-4 are well characterized structurally similar class A β-lactamases, which provide bacteria with resistance to penicillin-type antibiotics. Sharing 40% sequence identity, these β-lactamases were used to create a series of TEM-1/PSE-4 chimeras using structurebased recombination[1]. This project aims to understand the effect of this recombination on the properties of the resulting artificially-evolved enzymes. Specifically, we investigated whether the kinetic, structural and dynamic properties of TEM-1 and PSE-4 are conserved in different functional chimeras sharing between 64% to 94% sequence identity to TEM-1. Chimeras cTEM17m and cTEM-19m (respectively having 94% and 92% sequence identity to TEM-1) differ by only two substitutions (Met68 and Met69) neighbouring the catalytic nucleophile Ser70. The turnover rate of cTEM17m is native-like, indicating that its 17 mutations are functionally tolerated. However, the additional 2 mutations of cTEM-19m decreased the turnover rate by more than 20-fold relative to cTEM-17m. In order to isolate the energetic contribution these two substitutions, as well as to probe for synergistic or additive effects, we generated and kinetically characterized deconvolution mutants in both the chimeric and wild-type TEM-1 contexts. Double mutant cycle analysis revealed that the immediate neighbour of the catalytic Ser70, Met69, was the main contributor to decreased turnover rate. High resolution crystal structures of the chimera cTEM-19m and a deconvoluted mutant were obtained to verify the structural impact of the Met69 substitution. The overall structure of the chimeras and the deconvoluted mutant were similar to the parents. However, two structures of the chimera obtained in similar crystallisation conditions showed different active site conformations, suggesting increased molecular dynamics. NMR relaxation experiments on a fast (ps-ns) and slow (µsms) time scales confirmed broadly distributed new molecular dynamics in the chimera cTEM-17m that were not observed in either the wild-type TEM-1 or PSE-4 β-lactamases. The characterization of these artificially-evolved chimeras demonstrated dynamic states tolerated by β-lactamases, maintaining their functionality and potentially facilitating their evolution with regard to hydrolysis of new β-lactam compounds. [1] Nat Struct Biol. 9(7):553-8. Research funding provided by NSERC/CRSNG

Type de document: Document issu d'une conférence ou d'un atelier
Informations complémentaires: Affiche scientifique 89 Protein Science 21(suppl. 1):86
Mots-clés libres: -
Centre: Centre INRS-Institut Armand Frappier
Date de dépôt: 28 janv. 2024 15:00
Dernière modification: 28 janv. 2024 15:00
URI: https://espace.inrs.ca/id/eprint/14124

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