Combination of the mutations for improving activity of TEV protease in inclusion bodies

Hu, Jiong; Chen, Yinghua; Ren, Yuanyuan; Xiao, Wen‐Jing; Hu, Yafang; Yu, Xuelian; Fan, Jun

doi:10.1007/s00449-021-02589-5

Cited by 2 publications

(3 citation statements)

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“…Its applications have spanned various fields, including protein purification, protein interaction studies, protein variant generation, and therapeutic agent development [ 3 ]. However, its relatively slow catalytic rate poses a notable limitation [ 12 , 14 , 16 , 17 , 21 , 22 ]. To date, some successful examples mainly focus on directed evolution.…”

Section: Discussionmentioning

confidence: 99%

“…A combination of mutations TEVp5M (T17S, L56V, N68D, I77V, and S135G) [ 23 ] identified by rational design [ 14 ] and high-throughput screening [ 12 ], exhibited the highest solubility and slightly elevated catalytic activity in vivo. Fan et al found the TEVp5M-E106G variant enhanced the soluble production and cleavage activity of TEVp constructs [ 21 ]. The TEV-EAV variant (G79E,T173A) was introduced by YESS of combinatorial libraries [ 22 ], which retained high catalytic turnover.…”

Section: Discussionmentioning

confidence: 99%

“…The mutations of eTEV and uTEV3 extensively influence the structure, especially the S3I and P8Q of eTEV showed different connection point network characteristics compared with WT ( Figure 7 ). In addition, we found some reported mutated residues associated with enzyme activity on these pathways, such as T17 [ 14 ] and E106 [ 21 ]. The role of residues on these communication pathways will be further explored in our future research.…”

Section: Discussionmentioning

confidence: 99%

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Mechanism of Mutation-Induced Effects on the Catalytic Function of TEV Protease: A Molecular Dynamics Study

Wang,

Xu,

Wang

et al. 2024

Molecules

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Tobacco etch virus protease (TEVp) is wildly exploited for various biotechnological applications. These applications take advantage of TEVp’s ability to cleave specific substrate sequences to study protein function and interactions. A major limitation of this enzyme is its relatively slow catalytic rate. In this study, MD simulations were conducted on TEV enzymes and known highly active mutants (eTEV and uTEV3) to explore the relationship between mutation, conformation, and catalytic function. The results suggest that mutations distant from the active site can influence the substrate-binding pocket through interaction networks. MD analysis of eTEV demonstrates that, by stabilizing the orientation of the substrate at the catalytic site, mutations that appropriately enlarge the substrate-binding pocket will be beneficial for Kcat, enhancing the catalytic efficiency of the enzyme. On the contrary, mutations in uTEV3 reduced the flexibility of the active pocket and increased the hydrogen bonding between the substrate and enzyme, resulting in higher affinity. At the same time, the MD simulation demonstrates that mutations outside of the active site residues could affect the dynamic movement of the binding pocket by altering residue networks and communication pathways, thereby having a profound impact on reactivity. These findings not only provide a molecular mechanistic explanation for the excellent mutants, but also serve as a guiding framework for rational computational design.

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