Relevance of Local Flexibility Near the Active Site for Enzymatic Catalysis: Biochemical Characterization and Engineering of Cellulase Cel5A From Bacillus agaradherans

Abstract

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 Cel5A are selected for protein engineering via site-directed mutagenesis. Three Cel5A variants are 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 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.

Engineering Robust Cellulases for Tailored Lignocellulosic Degradation Cocktails

“…Another target area is the catalytic site, the targeting non-catalytic amino acids achieved a 1.9-fold improvement in the catalytic efficiency in an endoglucanase [79]. In addition, loops and residues that may interact with the substrate have been widely studied [80][81][82][83]. As a semi-rational approach also focused on protein areas that interact with the substrate, strategies employed a complete diversity for selected positions.…”

Rational Engineering ofBacillus cereusLeucine Dehydrogenase Towards α-keto Acid Reduction for Improving Unnatural Amino Acid Production

“…Substitution of T83A and T45M led to an additional hydrogen bond between Tyr112 and Thr114 on β5 fold which located in substrate entrance tunnel. Hydrogen bond network is very important as it is reported that disrupting a hydrogen bond network in the vicinity of the active site increases local flexibility of cellulase Cel5A which led to increased catalytic activity at low temperatures but sharply declined catalytic activity at high temperature . Enhancement of β5 fold stability by new formed hydrogen bond could improve the thermostability of Bc LeuDH, because the ridigity of β5 fold was important for substrate entrance and also Asp117 on β5 fold is thought to be functioned during catalysis .…”

Hot spots-making directed evolution easier

“…Three specific positions were selected based on the analysis of hydrogen bond patterns between residues within the active site. After sitedirected mutagenesis, Cel5A variants showed a concomitant increase in the catalytic activity at low temperatures and a decrease in activation energy and activation enthalpy, similar to cold-active enzymes, indicating that disrupting a hydrogen bond network in the vicinity of the active site increases local flexibility (Saavedra et al, 2018).…”