Pontificia Universidad Católica de Chile Pontificia Universidad Católica de Chile
Saavedra J.M., Azócar M.A., Rodríguez V., Ramírez-Sarmiento C., Andrews B.A., Asenjo J.A. and Parra L.P. (2018)

Relevance of local flexibility near the active site for enzymatic catalysis: Biochemical characterization and engineering of cellulase Cel5A from Bacillus agaradherans

Revista : Biotechnology Journal
Volumen : 13
Número : 8
Páginas : 10pp
Tipo de publicación : ISI Ir a publicación


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, we used cellulase Cel5A from Bacillus agaradherans as a model for in silico comparative analysis with mesophilic and psychrophilic counterparts. Structural features that determine flexibility were related to kinetic and thermodynamic parameters of catalysis. As a result, three specific positions in the vicinity of the active site of Cel5A were selected for protein engineering via site-directed mutagenesis. Three Cel5A variants were generated, N141L, A137Y and I102A/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 were 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. Our results provide a structural framework for explaining the changes in thermodynamic parameters observed between homologous enzymes with varying temperature adaptations.