Activation of Wild-Type Cytochrome P450BM3 by the Next Generation of Decoy Molecules: Enhanced Hydroxylation of Gaseous Alkanes and Crystallographic Evidence

Abstract

The direct hydroxylation of alkanes under mild conditions is a key issue in catalytic chemistry that addresses an increasing number of industrial and economic requirements. Cytochrome P450s are monooxygenases that are capable of oxidizing less reactive C−H bonds; however, wild-type P450s are unavailable for many important nonnative substrates such as gaseous alkanes. Here, we report the enhanced hydroxylation activities and crystallographic evidence for the role of decoy molecules in wild-type P450BM3-catalyzed hydroxylation of gaseous ethane and propane by using the next generation of decoy molecule. A cocrystal structure of P450BM3 and a decoy molecule reveals that an N-perfluoroacyl amino acid (decoy molecule) partially occupies the substrate-binding site of P450BM3. This binding of the decoy re-forms the active site pocket to allow the accommodation of small substrates and simultaneously influences the formation of compound I species by expelling water molecules from the active site.

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

“…[9] Fore xample,p ropane was efficiently hydroxylated by P450BM3 with the assistance of N-perfluorononanoyl-l-leucine (PFC9-l-Leu). [9] Fore xample,p ropane was efficiently hydroxylated by P450BM3 with the assistance of N-perfluorononanoyl-l-leucine (PFC9-l-Leu).…”

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 catalytic turnover numbers were found to be dependent on the chain length of the PFCs. Although ethane was hydroxylated under high pressure with PFCs, and at normal pressure with amino acid‐modified PFCs (PFCs‐amino acids), the current system was not able to oxidize methane …”

Catalytic Aerobic Oxidation of C(sp3)−H Bonds

“…[155] Hydroxylation rates of,r espectively 45 min À1 P450 À1 and 256 min À1 P450 À1 could be achieved with N-perfluorononanoyl-l-leucine. Theb inding of as ubstrate activates the enzyme to perform the oxidation reaction, which in itself can be quite aselective.Byadding an inert decoy molecule that binds and thereby activates the enzyme,c atalytic turnover for an onnative non-binding substrate might be achieved.…”