Enzyme Cold Adaptation through Evolution of Protein Flexibility

What lives, evolves. Macromolecular catalysis is a process, central to both evolutionary and metabolic aspect of life, as it provides a systematic bias favouring certain chemical processes. In living organisms, this bias is crucial for maintenance of constant composition outside of equilibrium with the environment, and transfer of hereditary information. Biochemical catalysis is based on facilitating the formation of the least probable state along the reaction pathway and is achieved by providing a suitable environment for that. High conformational versatility of their protein scaffold is the main reason why the enzymes perform a predominant part of cellular catalytic activity. Cold ecosystems pose a hard evolutionary challenge to the organisms populating them due to significant enzymatic rate retardation with decreasing temperature. At the same time, selective pressure for protein thermal stability is generally relieved in those organisms. Compared to their mesophilic and thermophilic (warm- and heat-active) counterparts, psychrophilic (cold-active) enzymes typically show a trade-off between activity and stability, with lower melting temperatures, but more favourably distributed activation parameters. As a decreased activation enthalpy and a more negative activation entropy enable a lower free energy penalty at low temperatures, the cold-active enzymes lose significantly less catalytically activity there. Such redistribution of thermodynamic parameters is generally attributed to the favourable dynamic patterns associated with a higher flexibility of the less ordered protein regions. In the present thesis, two different strategies of attaining enzyme psychrophilicity were explored. In our comparison of aliphatic tripeptide substrates breakdown by cold adapted salmon pancreatic elastase and its mesophilic ortholog, a significant shift between activation entropy and enthalpy of around 10 kcal/mol was observed. Notably, the structure of the psychrophilic elastase was also found to be more flexible at the protein surface. Our calculations have shown that the mutants of psychrophilic elastase, including certain mesophilic structural features in the surface loops, adopt significantly less cold-active character. The effect of these features, however, does not to appear to be simply cumulative. Our investigation of the orthologous pair of starch-degrading α-amylases revealed the psychrophilic ortholog originating from Antarctic bacteria only partly relies on redistribution of activation parameters to achieve favourable rates at low temperature. Curiously, this enzyme’s activity was found to be deteriorating at the temperatures significantly lower than the enzyme’s melting temperature. The computational reproduction of the temperature optimum for cold-active α-amylase enabled us to relate this phenomenon to a deterioration of prominent substrate stabilizing interaction with increasing temperatures of the environment. We provided a simple kinetic model incorporating two reactant states in equilibrium, where only the properly stabilized reactant state is catalytically active.Note that the manuscript I added manually is listed twice. First listing has the names of authors switched with their surnames. I am asking for that entry to be removed.