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.
Given prominent physicochemical similarities between
H2O2 and water, we report a new strategy for
promoting the
peroxygenase activity of P450 enzymes by engineering their water tunnels
to facilitate H2O2 access to the heme center
buried therein. Specifically, the H2O2-driven
activities of two native NADH-dependent P450 enzymes (CYP199A4 and
CYP153A
M.aq
) increase significantly (by
>183-fold and >15-fold, respectively). Additionally, the amount
of
H2O2 required for an artificial P450 peroxygenase
facilitated by a dual-functional small molecule to obtain the desired
product is reduced by 95%–97.5% (with ∼95% coupling
efficiency). Structural analysis suggests that mutating the residue
at the bottleneck of the water tunnel may open a second pathway for
H2O2 to flow to the heme center (in addition
to the natural substrate tunnel). This study highlights a promising,
generalizable strategy whereby P450 monooxygenases can be modified
to adopt peroxygenase activity through H2O2 tunnel
engineering, thus broadening the application scope of P450s in synthetic
chemistry and synthetic biology.
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