Comparing and Combining Alternative Strategies for Enhancing Cytochrome P450 Peroxygenase Activity

Cytochrome
P450 monooxygenase (CYP) enzymes have advantageous properties
over chemical catalysts. However, it is often not feasible to use
CYPs in larger-scale synthesis as they require additional cofactors
(NAD(P)H) and electron transfer proteins. This could be overcome by
transforming CYPs into peroxygenases that use H2O2. Recently, multiple strategies have been reported for converting
CYPs into peroxygenases. Mutating the residues of the acid–alcohol
pair in the oxygen-binding groove to those found in natural peroxygenases
can promote the desired H2O2-driven activity.
Another strategy is to enlarge the enzyme’s solvent channels
to allow H2O2 easier access into the active
site, to enhance peroxygenase activity. Here, we evaluate these different
strategies by comparing the peroxygenase activities of the double
I-helix mutant D251Q/T252E (the QE mutant) and the F182A mutant of
the bacterial enzyme CYP199A4. We also assess whether the peroxygenase
activity can be further improved by combining these mutations (to
give the F182AQE mutant). The F182A mutant exhibited the highest activity
toward a selection of smaller substrates that undergo O-demethylation, S-oxidation, and epoxidation reactions.
All the mutants converted 4-vinylbenzoic acid into the (S)-epoxide, with the F182A mutant having the highest stereoselectivity
(>99% ee). The F182A mutant was unable to oxidize 4-t-butylbenzoic acid, while the F182AQE mutant could with high activity.
The F182A mutation was found to substantially alter the selectivity
of the reaction with 4-ethylbenzoic acid, increasing hydroxylation
activity over desaturation. The F182A mutant catalyzed significant
further oxidation reactions of the primary metabolites before all
the substrate had been consumed, demonstrating a relaxed substrate
specificity. X-ray crystal structures of the F182A and F182AQE mutants
with the substrates revealed changes in substrate binding and solvent
access providing insights into these experimental observations.