Peroxide-Driven Hydroxylation of Small Alkanes Catalyzed by an Artificial P450BM3 Peroxygenase System

Abstract

We report the selective hydroxylation of small alkanes with H2O2 catalyzed by an artificial P450 peroxygenase system generated from engineered cytochrome P450BM3 variants in assistance with dual-functional small molecule (DFSM), in which DFSM acts as a general acid–base co-catalyst for activating H2O2. This peroxygenase system exhibited comparable catalytic turnover number (TON) to the fungal peroxygenase AaeUPO, the only known H2O2-dependent natural alkane hydroxylase. Moreover, when compared with evolved/engineered NADPH-dependent P450 variants, the current system yielded similar or even better product formation rates (PFRs) but lower total TONs. The substitution of the highly conserved T268 with amino acids having hydrophobic side chains was identified to play critical roles in improving the hydroxylation activity of the DFSM-facilitated P450BM3 peroxygenase system, which is distinct from NADPH-dependent P450 enzymes. These results offer useful insights into how to tune the catalytic functions and chemistry of P450 peroxygenases.

Harnessing P450 Enzyme for Biotechnology and Synthetic Biology

“…The terminal catalytical group played similar role of the engineered acid‐based residual through enzyme mutagenesis, [77] but showed more significant effect on the improvement of enzymatic turnover rate and enantiomeric specificity, [25] which was attributed to the more suitable position of the catalytic group of DFSM in the active site (Figure 9 A). Impressively, combined with enzyme mutagenesis, the DFSM based artificial P450BM3 peroxygenase has been successfully applied to oxidation of small alkenes which are with inert C−H bonds showing comparable turnover number to the H 2 O 2 ‐dependant natural alkane hydrolase [76] . In more recent studies, the effect of DFSM ( i. e ., Im‐C6‐Phe) on P450BM3 enzyme activity and enantioselectivities to synthesize ( R )‐styrene oxide has been further optimized by combination with enzyme engineering [78] .…”

Engineering cytochrome P450 enzyme systems for biomedical and biotechnological applications

“…The rate of epoxidation of (R)-(ϩ)-styrene and the enantiomeric specificity (ee value) of the product were dramatically increased (to ee 91%) by this innovative substrate engineering approach. This engineered peroxide-driven P450 BM3 system was further utilized to hydroxylate small alkanes with the assistance of Im-C6-Phe (118) (Fig. 3, compound 28).…”

Hemoprotein Catalyzed Oxygenations: P450s, UPOs, and Progress toward Scalable Reactions

“… 17 More recently, using P450BM3, Nguyen and co-workers created a nine-point mutant for the 4’-hydroxylation of atorvastatin using only H 2 O 2 , 81 although interestingly, the whole P450BM3 protein outperformed the heme domain in isolation. Further developments to the P450BM3 system for peroxide driven oxygenations employed a “dual function small molecule” (DFSM) approach, 82 in which N -(w-imidazol-1-yl hexanoyl)- l -phenylalanine (Im-C6-Phe) is bound in the P450 active site so as to provide the imidazole side-chain as an acid–base catalyst, designed to mimic a homologous glutamate in UPOs, which use H 2 O 2 as their natural route to Compound I. When Im-C6-Phe was included with mutant F87A/T268I/A184I in a biotransformation of propane, turnovers were 2-fold higher than had been recorded for UPOs with that substrate.…”