Optimization to low temperature activity in psychrophilic enzymes

Abstract

Psychrophiles, i.e., organisms thriving permanently at near-zero temperatures, synthesize cold-active enzymes to sustain their cell cycle. These enzymes are already used in many biotechnological applications requiring high activity at mild temperatures or fast heat-inactivation rate. Most psychrophilic enzymes optimize a high activity at low temperature at the expense of substrate affinity, therefore reducing the free energy barrier of the transition state. Furthermore, a weak temperature dependence of activity ensures moderate reduction of the catalytic activity in the cold. In these naturally evolved enzymes, the optimization to low temperature activity is reached via destabilization of the structures bearing the active site or by destabilization of the whole molecule. This involves a reduction in the number and strength of all types of weak interactions or the disappearance of stability factors, resulting in improved dynamics of active site residues in the cold. Considering the subtle structural adjustments required for low temperature activity, directed evolution appears to be the most suitable methodology to engineer cold activity in biological catalysts.

Keywords: biotechnology; cold adaptation; enzyme activity; extremophiles; psychrophiles.

Figure 1

Temperature dependence of activity. The activity of psychrophilic (open symbols, blue lines) and mesophilic (closed symbols) enzymes recorded at various temperatures illustrates the main properties of cold-adapted enzymes: cold activity and heat lability. Left panel, -amylases; right panel, cellulases. Both psychrophilic enzymes are from the Antarctic bacterium Pseudoalteromonas haloplanktis. Adapted from [11,12].

Figure 2

Inactivation and unfolding of psychrophilic enzymes. The activity of psychrophilic enzymes (upper panel, blue line) is inactivated by temperature before unfolding of the protein structure (lower panel, blue line) illustrating the pronounced heat-lability of the active site. By contrast, inactivation of mesophilic enzymes (black curves) closely corresponds to the loss of the protein conformation. Adapted from [14].

Figure 3

Structure of the active site. Superimposition of the active site residues in psychrophilic (blue) and mesophilic α-amylases (red). The chloride and calcium ions are shown as blue and green spheres, respectively. The 24 residues performing direct or water-mediated interactions with the substrate analog derived from acarbose (yellow) are identical and superimpose perfectly within the resolution of the structures, demonstrating a structural identity in these psychrophilic and mesophilic enzymes [27].

Figure 4

Optimization of activity by decreasing substrate affinity in psychrophilic enzymes. Reaction profile for an enzyme-catalyzed reaction with Gibbs energy changes under saturating substrate concentration. Weak substrate binding (in blue) decreases the activation energy (ΔG^#psychro) and thereby increases the reaction rate. In this scheme, the energy levels of E + S and of ES^# are assumed to be similar [10].