Direct Hydroxylation of Benzene to Phenol by Cytochrome P450BM3 Triggered by Amino Acid Derivatives

Abstract

The selective hydroxylation of benzene to phenol, without the formation of side products resulting from overoxidation, is catalyzed by cytochrome P450BM3 with the assistance of amino acid derivatives as decoy molecules. The catalytic turnover rate and the total turnover number reached 259 min P450BM3 and 40 200 P450BM3 when N-heptyl-l-proline modified with l-phenylalanine (C7-l-Pro-l-Phe) was used as the decoy molecule. This work shows that amino acid derivatives with a totally different structure from fatty acids can be used as decoy molecules for aromatic hydroxylation by wild-type P450BM3. This method for non-native substrate hydroxylation by wild-type P450BM3 has the potential to expand the utility of P450BM3 for biotransformations.

Biocatalytic Oxidation Reactions: A Chemist's Perspective

“…In addition to protein engineering,reaction and substrate engineering also efficiently modulate the reactivity and selectivity of P450 monooxygenases.D ecoy molecules,f or example,a re experiencing some interest in facilitating the conversion of small substrates. [22] Watanabe,R eetz, and others have reported that perfluorinated fatty acids can accelerate P450BM3-catalysed oxyfunctionalisation reactions of small aromatics, [23] short-chain alkanes, [24] and even methane. [25] Ther ationale behind these results is that the inert decoy compounds partially fill up the spacious active site of the enzyme and thereby facilitate the orientation of the small substrate to the heme active site.…”

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

“…Crystallography studies have revealed the role of decoy molecules in reshaping the active site of P450s. The co‐crystal structures of NADPH‐dependent P450BM3 bound to decoy molecules clearly indicate that perfluorononanoic acyl l ‐phenylalanine (PFC9‐ l ‐Trp) and Z‐ l ‐Pro‐ l ‐Phe partially occupy the native substrate tunnel and form a new pocket of reduced size (Figure A, B). This pocket allows the enzyme to accept smaller substrates, such as small alkanes and benzene.…”

“…The direct hydroxylation of benzene by P450BM3 exclusively gave phenol, with a product formation rate of 120 min −1 per P450 in the presence of perfluorononanoic acid (PFC9) or perfluorodecanoic acid (PFC10) . More recently, Watanabe and co‐workers reported an improved P450BM3 decoy system for benzene hydroxylation, in which the structure of the decoy molecule was optimized . The product formation rate and the total turnover number reached 259 min −1 per P450 and 40 200 per P450, respectively, when N ‐heptyl‐ l ‐proline‐ l ‐phenyl alanine (C7‐ l ‐Pro‐ l ‐Phe) was used as the decoy molecule .…”

“…More recently, Watanabe and co‐workers reported an improved P450BM3 decoy system for benzene hydroxylation, in which the structure of the decoy molecule was optimized . The product formation rate and the total turnover number reached 259 min −1 per P450 and 40 200 per P450, respectively, when N ‐heptyl‐ l ‐proline‐ l ‐phenyl alanine (C7‐ l ‐Pro‐ l ‐Phe) was used as the decoy molecule . Very recently, the same research group reported the first system for P450‐based whole‐cell biotransformation of benzene to phenol .…”

Crystals in Minutes: Instant On‐Site Microcrystallisation of Various Flavours of the CYP102A1 (P450BM3) Haem Domain

“…Decoy molecules, akin to the native substrate, bind to the substrate‐binding site of P450BM3, thereby stimulating the formation of the active oxygen species (Compound I, Cpd I; Figure bottom, red dotted box) without being hydroxylated themselves. Cpd I can then be used to hydroxylate non‐native compounds that fit into the space shaped by Cpd I and the decoy molecule, including ethane, propane, cyclohexane, and benzene . A comprehensive and in‐depth review regarding decoy molecules by Shoji et al can be found in ref.…”