An Inexpensive, Efficient Alternative to NADPH to Support Catalysis by Thermostable Cytochrome P450 Enzymes

Abstract

AbstractCytochrome P450 enzymes (P450s) are highly desirable catalysts for the regio‐ and stereo‐selective, late‐stage functionalization of pharmaceuticals and other fine chemicals. Recently, the resurrected ancestors of drug‐metabolizing P450s were shown to be highly thermostable and expressed in high yield, while retaining similar substrate specificity to the extant forms. However, they still rely on NADPH and cytochrome P450 reductase (CPR) to enable catalysis of oxidative transformations. To identify an alternative support system, we screened 10 oxygen surrogates (OSs) for the ability to support P450 ancestors from three different families. Of the 23 ancestors examined, 17 were supported by at least one OS as well as, or better than, by CPR. Using two candidate P450s we showed that OS‐dependent P450 catalysis can be optimized in a few steps, boosting product yield from ∼2.2 % with CPR to 88–100 % with an OS. The principles applied here will facilitate faster evaluation and optimization of OS‐supported P450 catalysis versus redox partner‐dependent P450 catalysis.