Acetate anion-triggered peroxygenation of non-native substrates by wild-type cytochrome P450s

Abstract

Cytochrome P450SPα (P450SPα) and cytochrome P450BSβ (P450BSβ) belonging to the CYP152 family of enzymes (CYP152s) can utilize H2O2 efficiently as an oxidant for the generation of compound I. Although P450SPα and P450BSβ have very high substrate specificity and catalyse hydroxylation of long-chain fatty acids exclusively, we found that they can oxidize non-native substrates such as styrene simply by including medium chain length n-alkyl carboxylic acids as "decoy molecules." Although we had assumed that acetic acid did not serve as a decoy molecule, P450SPα and P450BSβ efficiently catalysed oxidation of non-native substrates when the reaction was carried out at a high concentration of acetate anion. The turnover rate for epoxidation of styrene catalysed by P450BSβ in the presence of 1 M acetate anion reached 590 ± 30 min(-1).

A substrate-binding-state mimic of H2O2-dependent cytochrome P450 produced by one-point mutagenesis and peroxygenation of non-native substrates

“…That no catalytic activity was observed with R241E indicates that the carboxylate of R241E is also too far from the heme iron. The stereoselectivity of styrene epoxidation by mutants was (S)-configuration, whereas the wild-type P450 SPα in the presence of acetic acid gave the (R)-isomer, 33 suggesting that the replacement of Ala-245 affects the styrene orientation in the active site. We also examined the hydroxylation of indole to give indigo 45 as well as the hydroxylation of 1-methoxynaphthalene to give Russig's blue 31 (Scheme S1 †) and found that the A245E mutant also catalyzes these reactions.…”

New function of aldoxime dehydratase: Redox catalysis and the formation of an expected product

“…To measure peroxygenase activity, we used 1-methoxynaphthalene (1-MN) as a substrate, and measured the absorbance at 610 nm due to the absorption maximum of the reaction product (Russig’s blue) [46, 47] [Fig 4A(a)]. In the reaction mixture containing 5 mM H 2 O 2 , 0.5 mM 1-MN and 1 μM OxdA, the marked production of Russig’s blue was observed under the standard assay D conditions (S9 Fig).…”

“…The reaction was started by the addition of the enzyme and was carried out for 1 min at 30°C. The production of Russig’s blue, which is a reaction product, was determined from the increase in the absorbance at 610 nm [(ε) 1.45 × 10 4 M -1 cm -1 ] [46, 47]. One unit of peroxygenase activity was defined as the amount of enzyme that catalyzed the consumption of 0.25 μmol of Russig’s blue/min (equal to the consumption of 1 μmol of H 2 O 2 /min) under the standard assay D conditions.…”

Strategies for Substrate‐Regulated P450 Catalysis: From Substrate Engineering to Co‐catalysis

“…Interestingly, Watanabe et al. recently demonstrated that acetate anions at high concentration can also serve as decoy molecules to efficiently epoxidize styrene with P450 BSβ . Furthermore, the decoy molecule strategy was also applied to the H 2 O 2 ‐dependent cytochrome P450 spα enzyme CYP152B1, which was used in stereoselective epoxidation with chiral ibuprofen as a decoy molecule (Table ) .…”