Computational thermostabilization of an enzyme

Abstract

Thermostabilizing an enzyme while maintaining its activity for industrial or biomedical applications can be difficult with traditional selection methods. We describe a rapid computational approach that identified three mutations within a model enzyme that produced a 10 degrees C increase in apparent melting temperature T(m) and a 30-fold increase in half-life at 50 degrees C, with no reduction in catalytic efficiency. The effects of the mutations were synergistic, giving an increase in excess of the sum of their individual effects. The redesigned enzyme induced an increased, temperature-dependent bacterial growth rate under conditions that required its activity, thereby coupling molecular and metabolic engineering.

Figure 1. Thermal denaturation and activity half-life measurements

A) Temperature melt measuring the change in signal at 220nm over a range of temperatures. All constructs show a folded baseline followed by a sigmodial two-state transition to an unfolded baseline. Only a blow-up of data from 40° to 70° is shown; at lower temperatures the baseline plateaus corresponding to an assignment of 100% folded protein. B) Activity decay at 50 °C. Wild-type yCD and the double and triple mutant constructs were incubated at 50° C and their activity was measured over time as described in methods. The resulting curves gave half-lives for the enzymes at 50° C of 4 hours for WT, 21 hours for the Double mutant and 117 hours for the triple mutant.

Figure 2. In vivo assay for metabolic growth phenotype

Bacterial growth curves in media conditions requiring cytosine deaminase activity for generation of uracil, as described in methods. Both wild-type and reengineered mutants of yCD complement the bacterial activity; the thermostabilized enzyme variant displays a slight increase in growth rate at 30 °C and a significant increase at 37°C.

Figure 3. Structural analyses

A) Van der Waals representation of residues Y19, A23, Y26 and I140 in the wild-type yCD crystal structure (left) and same representation and orientation for the mutant construct with A23L and I140L mutations (right). B) Van der Waals radii representation of the area around V108 in the wild-type structure (left) and similar representation of the area around the V108I mutation in the triple mutant crystal structure.