Hybrid Thermophilic/Mesophilic Enzymes Reveal a Role for Conformational Disorder in Regulation of Bacterial Enzyme I.
Bacterial Proteins
/ chemistry
Catalysis
Enzyme Activation
Enzyme Stability
Escherichia coli
/ enzymology
Firmicutes
/ enzymology
Models, Molecular
Phosphoenolpyruvate Sugar Phosphotransferase System
/ chemistry
Phosphotransferases (Nitrogenous Group Acceptor)
/ chemistry
Protein Conformation
Protein Domains
Protein Engineering
/ methods
Recombinant Fusion Proteins
/ chemistry
Thermodynamics
NMR relaxation
bacterial PTS
enzyme regulation
metadynamics
protein dynamics
Journal
Journal of molecular biology
ISSN: 1089-8638
Titre abrégé: J Mol Biol
Pays: Netherlands
ID NLM: 2985088R
Informations de publication
Date de publication:
24 07 2020
24 07 2020
Historique:
received:
16
04
2020
revised:
23
05
2020
accepted:
29
05
2020
pubmed:
7
6
2020
medline:
26
1
2021
entrez:
7
6
2020
Statut:
ppublish
Résumé
Conformational disorder is emerging as an important feature of biopolymers, regulating a vast array of cellular functions, including signaling, phase separation, and enzyme catalysis. Here we combine NMR, crystallography, computer simulations, protein engineering, and functional assays to investigate the role played by conformational heterogeneity in determining the activity of the C-terminal domain of bacterial Enzyme I (EIC). In particular, we design chimeric proteins by hybridizing EIC from thermophilic and mesophilic organisms, and we characterize the resulting constructs for structure, dynamics, and biological function. We show that EIC exists as a mixture of active and inactive conformations and that functional regulation is achieved by tuning the thermodynamic balance between active and inactive states. Interestingly, we also present a hybrid thermophilic/mesophilic enzyme that is thermostable and more active than the wild-type thermophilic enzyme, suggesting that hybridizing thermophilic and mesophilic proteins is a valid strategy to engineer thermostable enzymes with significant low-temperature activity.
Identifiants
pubmed: 32504625
pii: S0022-2836(20)30375-2
doi: 10.1016/j.jmb.2020.05.024
pmc: PMC7963438
mid: NIHMS1600622
pii:
doi:
Substances chimiques
Bacterial Proteins
0
Recombinant Fusion Proteins
0
Phosphoenolpyruvate Sugar Phosphotransferase System
EC 2.7.1.-
Phosphotransferases (Nitrogenous Group Acceptor)
EC 2.7.3.-
phosphoenolpyruvate-protein phosphotransferase
EC 2.7.3.9
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.
Langues
eng
Sous-ensembles de citation
IM
Pagination
4481-4498Subventions
Organisme : NIGMS NIH HHS
ID : P30 GM124169
Pays : United States
Organisme : NIGMS NIH HHS
ID : R35 GM133488
Pays : United States
Informations de copyright
Copyright © 2020 Elsevier Ltd. All rights reserved.
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