Journal
BIOTECHNOLOGY JOURNAL
Volume 13, Issue 8, Pages -Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/biot.201700669
Keywords
cellulase; flexibility; molecular dynamics; protein engineering
Funding
- CONICYT Basal Centre Grant for the Centre for Biotechnology and Bioengineering, CeBiB [FB0001]
- National Laboratory for High Performance Computing, NLHPC [ECM-02]
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Detailed molecular mechanisms underpinning enzymatic reactions are still a central problem in biochemistry. The need for active site flexibility to sustain catalytic activity constitutes a notion of wide acceptance, although its direct influence remains to be fully understood. With the aim of studying the relationship between structural dynamics and enzyme catalysis, the cellulase Cel5A from Bacillus agaradherans is used as a model for in silico comparative analysis with mesophilic and psychrophilic counterparts. Structural features that determine flexibility are related to kinetic and thermodynamic parameters of catalysis. As a result, three specific positions in the vicinity of the active site of CeI5A are selected for protein engineering via site-directed mutagenesis. Three CeI5A variants are generated, N141 L, A137Y and 1102A/A137Y, showing a concomitant increase in the catalytic activity at low temperatures and a decrease in activation energy and activation enthalpy, similar to cold-active enzymes. These results are interpreted in structural terms by molecular dynamics simulations, showing that disrupting a hydrogen bond network in the vicinity of the active site increases local flexibility. These results provide a structural framework for explaining the changes in thermodynamic parameters observed between homologous enzymes with varying temperature adaptations.
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