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.

Deuterium quadrupole splittings from two series of optical isomers, l- and d-N-acyl-1-phenyl-d 5-2-aminopropane, with linear acyl chains ranging from 1 to 10 carbon atoms, were measured using 2H NMR spectroscopy. Both series of molecules were dissolved in anionic nematic cholesteric lyotropic liquid crystals prepared with cesium N-dodecanoyl-l-alaninate. With these values, the two order parameters that completely characterize the average alignment of the aromatic ring were calculated. Differential incorporation of both series of isomers into the aggregate is observed for intermediate size molecules, as evidenced by the values of the order parameters of the ring. Our results indicate that the first two derivatives in both series, l- and d-C1 and l- and d-C2, are located near the interface, possibly forming a H bond between the NH and/or CO groups and the interstitial water molecules. Increasing the hydrophobicity of the chain by adding one carbon atom induces a decrease in the overall alignment. Intermediate acyl chain length molecules progressively incorporate sufficiently into the aggregate for both asymmetric carbons, from the surface and the guest molecule, to approach each other, introducing differences in the average alignment of the aromatic ring between both series of isomers. Longer acyl chain derivatives incorporate deeper into the micelle and do not show this differentiation, possibly because the asymmetric carbons are located far away from each other.