S6: Engineering Cytochrome P450 Enzymes for Biocatalysis

Cytochrome P450 monooxygenases (also known as CYP) constitute the largest family of heme-containing monooxygenases that can oxidize a broad range of substrates, often at non-reactive carbon centers.  Because of their unique chemistry and high selectivity, cytochrome P450 monooxygenases have been increasingly used as biocatalysts for synthesis of pharmaceutical intermediates.  Here I discuss our recent effort in exploring cytochrome P450 enzymes for biocatalysis.  In the first example, we used directed evolution to invert the enantioselectivity of a novel P450pyr enzyme from Sphingomonas sp. HXN-200.  Iterative targeted site-saturation mutagenesis coupled with a novel high-throughput two-enzyme-based colorimetric assay was used to create a P450pyr mutant that shows an ee of 83% (R) compared to the wild type’s ee of 43% (S).  The resulting mutant can be used to convert N-benzyl pyrrolidine to its corresponding (R)- and (S)-N-benzyl-3-hydroxypyrrolidines that are important pharmaceutical intermediates in the synthesis of a k-receptor agonist, an antibacterial agent, carbapenem antibiotics, and a 5-HT_1Da receptor agonist.  In the second example, we used a P450BM3 enzyme in combination with an organometallic complex to enable enantioselective epoxidation of a mixture of olefins or a single internal olefin to a terminal epoxide in an enantioenriched form.  The organometallic complex catalyzes the isomerization of the olefin to an equilibrium mixture of internal and terminal olefin, while the enzyme selectively reacts with the terminal olefin to form an epoxide.  Such a system takes the advantages of both enzyme catalysts and chemical catalysts.