A dynamical perspective on cold-adapted enzymes at the molecolar level

Some organisms, especially unicellular, have adapted to extreme temperature conditions, and are capable of proliferating in such environments mainly thanks to the optimisation of their enzymatic repertoire. Cold-adapted (psychrophilic) enzymes are invariably characterised by high catalytic activity at low temperatures, necessary to endure the exponential reduction of the speed of chemical reactions in these conditions, and by low thermal stability. Numerous studies aimed at understanding the mechanisms of such adaptation at the molecular level agree that the high turnover observed for psychrophilic enzymes results from a decrease of the activation enthalpy of the catalysed reaction, which in turn is achieved structurally by decreasing the number and strength of the enthalpically stabilised interactions which must be broken to reach the transition state. Since these interactions are also involved in the stabilisation of the enzyme structure, their decrease has the effect of increasing structural flexibility, as reflected by the low thermal stability found for psychrophilic enzymes. Flexibility seems thus to have a fundamental role in enzymatic cold adaptation, and its study by means of Molecular Dynamics (MD) simulations provides a detailed description taking rigorously into account its dynamical character. I report the results of comparative MD studies performed on homologous mesophilic and psychrophilic enzymes, and mutants thereof, to investigate the molecular bases of cold adaptation. Long, multi-replica and explicit-solvent MD simulations have been compared in particular in terms of molecular flexibility and the dynamics of intramolecular interactions. Results show that differences in the dynamic character of the compared enzymes are found in loops surrounding the active site or substrate-binding cleft. In the case of chloride-dependent alpha-amylases, the comparison of the cold-active enzyme from Pseudoalteromonas Haloplanktis with its mesophilic porcine homologue shows that modulation of the size and flexibility of these loops cause the immediate surroundings of the active site to be comparatively more flexible in the psychrophilic enzyme. Detailed analysis of these enzymes active-site dynamics reveals that elements previously identified through X-ray crystallography as involved in substrate binding in both enzymes undergo concerted motions that may be linked to catalysis. The comparison of psychrophilic and mesophilic isoforms of trypsin from Salmo Salar shows that the cold-adapted enzyme presents fewer interdomain interactions and enhanced localised flexibility in regions close to the catalytic site. Notably, these regions fit well with the pattern of protein flexibility previously reported for psychrophilic elastases. These results indicate that specific sites within the serine-protease fold can be considered hot spots of cold-adaptation and that psychrophilic trypsins and elastases have independently discovered similar molecular strategies to optimise flexibility at low temperatures. This evidence of evolutionary convergence underlines the importance of extending intrafamiliar comparative studies to unveil general features of how enzymes adapt their dynamical properties low temperatures. Molecular dynamics studies using all-atomic models, as those presented herein, have proven their effectiveness in this context, but their computational requirement hampers their applicability as the size of the test set increases. For these reasons it would be relevant to devise a simplified ("coarse-grain") approach to perform comparative analyses at a higher throughput than is currently feasible. To evaluate the level of simplification most suitable for this application, a "coarse-grain" approach has been adopted to study the mechanical properties of trypsins, taking into account results obtained using all-atomic models. The results of these comparisons are driving the development of a multi-scale "coarse-grain" approach that combines the required efficiency and accuracy.