DOI: 10.1073/pnas.1812204115
论文题名: Exploiting correlated molecular-dynamics networks to counteract enzyme activity–stability trade-off
作者: Yu H. ; Dalby P.A.
刊名: Proceedings of the National Academy of Sciences of the United States of America
ISSN: 0027-8424
出版年: 2018
卷: 115, 期: 52 起始页码: E12192
结束页码: E12200
语种: 英语
英文关键词: Dynamics
; Protein engineering
; Stability
; Trade-off
; Transketolase
Scopus关键词: aldehyde
; dimer
; transketolase
; aldehyde
; Escherichia coli protein
; transketolase
; Article
; correlational study
; enzyme active site
; enzyme activity
; enzyme kinetics
; enzyme stability
; enzyme structure
; enzyme substrate
; Escherichia coli
; molecular dynamics
; nonhuman
; priority journal
; protein aggregation
; protein expression
; protein unfolding
; simulation
; thermodynamics
; thermostability
; wild type
; chemistry
; enzyme specificity
; enzyme stability
; enzymology
; genetics
; kinetics
; metabolism
; molecular dynamics
; protein conformation
; protein engineering
; Aldehydes
; Catalytic Domain
; Enzyme Stability
; Escherichia coli
; Escherichia coli Proteins
; Kinetics
; Molecular Dynamics Simulation
; Protein Conformation
; Protein Engineering
; Substrate Specificity
; Transketolase
英文摘要: The directed evolution of enzymes for improved activity or substrate specificity commonly leads to a trade-off in stability. We have identified an activity–stability trade-off and a loss in unfolding cooperativity for a variant (3M) of Escherichia coli transketolase (TK) engineered to accept aromatic substrates. Molecular dynamics simulations of 3M revealed increased flexibility in several interconnected active-site regions that also form part of the dimer interface. Mutating the newly flexible active-site residues to regain stability risked losing the new activity. We hypothesized that stabilizing mutations could be targeted to residues outside of the active site, whose dynamics were correlated with the newly flexible active-site residues. We previously stabilized WT TK by targeting mutations to highly flexible regions. These regions were much less flexible in 3M and would not have been selected a priori as targets using the same strategy based on flexibility alone. However, their dynamics were highly correlated with the newly flexible active-site regions of 3M. Introducing the previous mutations into 3M reestablished the WT level of stability and unfolding cooperativity, giving a 10.8-fold improved half-life at 55 °C, and increased midpoint and aggregation onset temperatures by 3 °C and 4.3 °C, respectively. Even the activity toward aromatic aldehydes increased up to threefold. Molecular dynamics simulations confirmed that the mutations rigidified the active-site via the correlated network. This work provides insights into the impact of rigidifying mutations within highly correlated dynamic networks that could also be useful for developing improved computational protein engineering strategies. © 2018 National Academy of Sciences. All rights reserved. Citation statistics:
资源类型: 期刊论文
标识符: http://119.78.100.158/handle/2HF3EXSE/163626
Appears in Collections: 气候变化与战略
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作者单位: Yu, H., Department of Biochemical Engineering, University College London, London, WC1H 0AH, United Kingdom; Dalby, P.A., Department of Biochemical Engineering, University College London, London, WC1H 0AH, United Kingdom
Recommended Citation:
Yu H.,Dalby P.A.. Exploiting correlated molecular-dynamics networks to counteract enzyme activity–stability trade-off[J]. Proceedings of the National Academy of Sciences of the United States of America,2018-01-01,115(52)