(511b) Engineering Robust Activity in Extremophilic Enzymes

Enzymes are highly specific and environmentally friendly biological catalysts with a potential multibillion dollar market. Examples of their applications include a wide array of industries, such as food and beverages, textiles, detergents, pharmaceuticals, and production of biofuels. The broad range of operating temperatures of these industries require the catalytic activity of enzymes to be less sensitive to temperature. Accordingly, the physical and chemical basis for the temperature adaptability of enzymes from psychrophiles and thermophiles are extensively studied. These studies indicate that the cold adaptability of psychrophilic enzymes is due to their high flexibility, specifically near their active site. In contrast, the loss of flexibility helps thermophilic enzymes to retain their stability at high temperatures.

We hypothesize that the incorporation of flexibility near the active site of thermophilic enzyme can increase its activity at low temperatures without compromising its overall stability. We test this hypothesis by mutating glutamic acid to glycine near the active site of Geo thermocatenulatus (GTL) – a thermophilic enzyme. We performed two such mutations, Glu316Gly and Glu361Gly and observed an increase in the specific activity at both lower and higher temperatures compared to the wild type (WT) GTL. We use all-atom molecular dynamics (MD) simulations to explore the local and global flexibilities and understand the mechanisms through which the mutations have increased the catalytic activity of GTL. In our presentation, we will discuss these results and comment on their implications for designing enzymes with a broader range of stability and activity.