Surface charged amino acid-based strategy for rational engineering of kinetic stability and specific activity of enzymes: Linking experiments with computational modeling
- Resource Type
- Authors
- Mingxue Yuan; Qingping Liang; Zhemin Liu; Haijin Mou; Xiaodan Fu; Changliang Zhu
- Source
- International journal of biological macromolecules. 182
- Subject
- Hot Temperature
Protein Conformation
Mutant
02 engineering and technology
Molecular Dynamics Simulation
Protein Engineering
Biochemistry
Catalysis
03 medical and health sciences
symbols.namesake
Structural Biology
Catalytic Domain
Enzyme Stability
Molecular Biology
030304 developmental biology
chemistry.chemical_classification
0303 health sciences
beta-Mannosidase
Substrate (chemistry)
General Medicine
Protein engineering
021001 nanoscience & nanotechnology
Amino acid
Gibbs free energy
Kinetics
Enzyme
chemistry
symbols
Biophysics
Thermodynamics
Specific activity
0210 nano-technology
- Language
- ISSN
- 1879-0003
A rational workflow for engineering kinetically stable enzymes with good specific activity by surface charged amino acids engineering was proposed based on systematically analyzing the results of mutating 44 negatively charged surface amino acids of a thermophilic β-mannanase (ManAK). Computational data, combined with experimental results indicated that percentage side-chain solvent accessibility (PSSA), changes in Gibbs free energy of unfolding (∆∆Gmut) and root-mean-square fluctuations (RMSF) could be suitable for screening kinetically stable mutants. A combinational standard (∆∆Gmut 0.68 A) resulted a decrease in the proportion of destabilizing mutants to 12.5%. The perturbations of substrate affinity and specific activity caused by mutation were weakened as the shortest distance from Cα of mutated site to Cα of catalytic sites (DsCα-Cα) increased. Results indicated that hotspot zones contributing to the local stability and integrity of catalytic motif at elevated temperatures might be widely distributed across spatial structure of the protein, while the mutation perturbation on enzyme specific activity demonstrated a gradually weakening trend from the catalytic core to the protein surface. These findings further our understanding of the structural-functional relationships of protein and highlight a deduced workflow to engineering industrially useful enzymes.