Mechanistic Insights into CYP199A4-Catalyzed α-Hydroxyketone Formation and Hydrogen Bond-Assisted C-C Bond Cleavage Catalyzed by the CYP199A4 F182L Mutant.

CYP199A4 is a cytochrome P450 and can catalyze the hydroxylation of 4-propionylbenzoic acid (4-pIBA) to generate α-hydroxyketone with high stereoselectivity. The F182L mutant of CYP199A4 (F182L-CYP199A4) has been shown to support the cleavage of the C-C bond between the carbonyl and hydroxyl groups of α-hydroxyketone, whereas wild-type CYP199A4 cannot. To uncover how the Phe182 regulates substrate reactivity, we conducted classical molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) MD simulations on these systems. The results predicted that the formation of α-hydroxyketone preferentially led to the (S)-enantiomer. Moreover, the findings revealed that the F182L-CYP199A4 facilitated the formation of a hydrogen bond between the α-hydroxyketone and the reactive peroxoanion (POA) species. This interaction stabilized the α-hydroxyketone near POA and promoted the subsequent C-C bond cleavage. The mechanism of α-hydroxyketone formation and the subsequent C-C bond cleavage were elucidated by employing the hybrid density functional theory (DFT). The α-hydroxyketone formation mechanism involved C-H hydroxylation of 4-pIBA with a rate-limiting energy barrier of 17.1 kcal/mol. The C-C bond cleavage of α-hydroxyketone catalyzed by F182L-CYP199A4 occurred via a radical attack mechanism.