Hoodwinking Cytochrome P450BM3 into Hydroxylating Non-Native Substrates by Exploiting Its Substrate Misrecognition

Area of Science:

• Biocatalysis and Synthetic Chemistry
• Enzyme Engineering and Directed Evolution

Background:

• Bacterial cytochrome P450s (P450s) exhibit high activity for C-H bond hydroxylation, making them promising biocatalysts for green chemistry.
• However, their narrow substrate specificity limits their application with non-native substrates.
• Mutagenesis is typically required to alter P450 activity, posing limitations for broader use.

Purpose of the Study:

• To develop a method for enabling P450BM3 to oxidize non-native substrates without mutagenesis.
• To design inert
• decoy molecules
• that mimic native substrates and activate P450BM3.
• To enhance catalytic activity and control selectivity for non-native substrate hydroxylation.

Main Methods:

• Design and synthesis of decoy molecules, including perfluorinated carboxylic acids (PFCs) and N-acyl amino acids.
• Crystallographic analysis of P450BM3 complexed with decoy molecules to elucidate binding modes.
• Assays to measure catalytic activity and stereoselectivity for non-native substrate hydroxylation.
• Whole-cell bioreactor experiments using E. coli expressing P450BM3.

Main Results:

• Decoy molecules, such as PFCs and N-acyl amino acids, successfully activated P450BM3 for non-native substrate oxidation.
• Structural studies revealed how decoy molecules bind in the active site, leaving space for non-native substrates.
• Third-generation decoy molecules, like N-enanthyl-l-proline modified with l-phenylalanine, significantly enhanced turnover rates and controlled hydroxylation stereoselectivity.
• Decoy molecules enabled P450BM3 to function in whole-cell biocatalysis and showed altered regioselectivity with Mn-substituted P450BM3.

Conclusions:

• Decoy molecules provide a powerful strategy to engineer the reactivity of wild-type P450BM3 for diverse non-native substrates.
• This approach expands the utility of P450 enzymes in green synthetic chemistry and whole-cell biocatalysis.
• The design of decoy molecules allows for fine-tuning of both catalytic activity and stereoselectivity, offering precise control over enzymatic reactions.