Extending the capabilities of nature's most versatile catalysts: directed evolution of mammalian xenobiotic-metabolizing P450s.

Cytochrome P450 enzymes are amongst the most versatile enzymatic catalysts known. The ability to introduce a single atom of oxygen into an organic substrate has led to the diversification and exploitation of these enzymes throughout nature. Nowhere is this versatility more apparent than in the mammalian liver, where P450 monooxygenases catalyze the metabolic clearance of innumerate drugs and other environmental chemicals. In addition to the aromatic and aliphatic hydroxylations, N- and O-dealkylations, and heteroatom oxidations that are common in drug metabolism, many more unusual reactions catalyzed by P450s have been discovered, including reductions, group transfers and other biotransformations not typically associated with monooxygenases. A research area that shows great potential for development over the next few decades is the directed evolution of P450s as biocatalysts. Mammalian xenobiotic-metabolizing P450s are especially well suited to such protein engineering due to their ability to interact with relatively wide ranges of substrates with marked differences in structure and physicochemical properties. Typical characteristics, such as the low turnover rates and poor coupling seen during the metabolism of xenobiotics, as well as the enzyme specificity towards particular substrates and reactions, can be improved by directed evolution. This mini-review will cover the fundamental enabling technologies required to successfully engineer P450s, examine the work done to date on the directed evolution of mammalian forms, and provide a perspective on what will be required for the successful implementation of engineered enzymes.