Controlling the Regio- and Stereoselectivity of Cytochrome P450 Monooxygenases by Protein Engineering
Cytochrome P450 monooxygenases (CYPs) have been used in synthetic organic chemistry and biotechnology for decades, especially in CH-activating oxidative hydroxylation. However, the control of both the regio- and stereoselectivity on a broad basis, a prerequisite for ecologically and economically viable transformations, has remained elusive until recent years, that have seen the advent of advanced methods in protein engineering. Successful examples of rational design utilizing site-specific mutagenesis have appeared, but the more general and reliable approach is directed evolution based on recursive cycles of mutagenesis, expression and screening. Since the screening step is the labor-intensive part (the bottleneck of directed evolution), advanced mutagenesis methods and strategies have been developed during the last 6–8 years. Saturation mutagenesis at multiple sites lining the binding pocket utilizing reduced amino acid alphabets and its iterative embodiment (ISM) have proven to be particularly effective, requiring minimal screening. The choice of amino acids as combinatorial building blocks is guided by structural, mechanistic, consensus and computational data. Directed evolution and rational design are beginning to merge for maximal efficiency.