Gobeil, Sophie; Park, Jaeok; Clouthier, Christopher M.; Doucet, Nicolas 
ORCID: 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,.
Résumé
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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