Investigation of cytochrome P450-catalyzed lyase and hydroxylation reactions with labeled peroxide

The heme enzymes of the cytochrome P450 superfamily (CYPs) catalyze oxidation reactions with a high level of selectivity. The most common reactions they catalyze are C-H bond hydroxylations but one of the important and more enigmatic reactions they can catalyze is the oxidative cleavage of C-C bonds of α-hydroxyketones, a crucial reaction in steroid biosynthesis. Variants of the CYP199A4 enzyme from the bacterium Rhodopseudomonas palustris HaA2 are used to catalyze both the hydroxylation and C-C bond cleavage reactions with designer substrate probes using hydrogen peroxide (H 2 O 2 ) as the oxidant. Both 4-propionyl- and 4-(2′-oxopropyl)-benzoic acids were regioselectively hydroxylated by the CYP199A4 enzyme to generate α-hydroxyketone metabolites (4-(2′-hydroxypropanoyl)benzoic acid and 4-(1′-hydroxy-2′-oxopropyl)benzoic acid, respectively). These α-hydroxyketones, along with 4-(1′-hydroxy-1′-methyl-2′-oxopropyl)benzoic acid (JCM 1) and 4-(1′-oxo-2′-hydroxy-2′-methylpropyl)benzoic acid (JCM 2) were synthesized and used as substrates with these peroxygenase variants of the CYP199A4 enzyme. Isotopically 18 O-labelled hydrogen peroxide was used to facilitate the identification of enzyme-generated metabolites by GC-MS. The F182A variant of CYP199A4 efficiently catalyzes C-C bond cleavage across these four substrate probes and shows activity analogous to that supported by the steroid-biosynthesizing CYP17 family, facilitating advanced mechanistic studies of CYP lyase activity.