Changing the substrate specificity of cytochrome c peroxidase using directed evolution.

Cytochrome c peroxidase (CCP) from Saccharomyces cerevisiae was subjected to directed molecular evolution to generate mutants with increased activity against 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). Using a combination of DNA shuffling and saturation mutagenesis, mutants were isolated which possessed more than 20-fold increased activity against ABTS and a 70-fold increased specificity toward ABTS compared to the natural substrate. In contrast, activities against another small organic molecule, guaiacol, were not significantly affected. Mutations at residues Asp224 and Asp217 were responsible for this increase in activity. These two residues are located on the surface of the protein and not in the direct vicinity of the distal cavity of the peroxidase, where small organic substrates are believed to be oxidized. Mutations at position Asp224 also lead to an increased amount of the active holoenzyme expressed in Escherichia coli, favoring the selection of these mutants in the employed colony screen. Possible explanations for the effect of the mutations on the in vitro activity of CCP as well as the increased amount of holoenzyme are discussed.

Directed molecular evolution of cytochrome c peroxidase.

Cytochrome c peroxidase (CCP) from Saccharomyces cerevisiae was subjected to directed molecular evolution to generate mutants with increased activity against the classical peroxidase substrate guaiacol, thus changing the substrate specificity of CCP from the protein cytochrome c to a small organic molecule. After three rounds of DNA shuffling and screening, mutants were isolated which possessed a 300-fold increased activity against guaiacol and an up to 1000-fold increased specificity for this substrate relative to that for the natural substrate. In all of the selected mutants, the distal arginine (Arg48), which is fully conserved in the superfamily of peroxidases, was mutated to histidine, showing that this mutation plays a key role in the significant increase in activity against phenolic substrates. The results suggest that, in addition to stabilizing the reactive intermediate compound I, the distal arginine plays an important role as a gatekeeper in the active site of CCP, controlling the access to the ferryl oxygen and the distal histidine. Other isolated mutations increase the general reactivity of the peroxidase or increase the intracellular concentration of the active holo form, allowing their selection under the employed screening conditions. The results illustrate the ability of directed molecular evolution technologies to deliver solutions to biochemical problems that would not be readily predicted by rational design.