Evolutionary Approaches to Study Cytochrome c Peroxidase

Abstract

Directed molecular evolution of enzymes and proteins has emerged as an extremely powerful method to create proteins with novel properties, both for practical applications as well as for mechanistic studies. To demonstrate the underlying principles of this approach, we describe here our work on the heme-containing cytochrome c peroxidase (CCP) from Saccharomyces cerevisiae. Using directed evolution, we changed the substrate specificity of CCP from the protein cytochrome c to a small organic molecule with the best mutants possessing up to 300-fold higher activity against a phenolic substrate. In addition to novel insights into the mechanism of peroxidases, 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.