Orthologue inference-based enzyme mining for diversification of the anti-cancer evodiamine scaffold

Area of Science:

• Biocatalysis
• Medicinal Chemistry
• Bioinformatics

Background:

• Selective functionalization of complex alkaloid scaffolds is crucial for drug discovery but chemically challenging.
• Cytochrome P450 monooxygenases offer a solution by enabling stereo- and regioselective C-H functionalization.

Purpose of the Study:

• To develop a targeted workflow for discovering novel enzymes (cytochrome P450s) capable of modifying alkaloid scaffolds.
• To leverage orthologue-inference bioinformatics for efficient enzyme mining from diverse plant species.

Main Methods:

• Comparative analysis of 15 alkaloid-producing plant species to generate a focused library of 15 P450 enzymes.
• Biocatalytic screening of the P450 library against alkaloid substrates.
• Structural modeling and site-directed mutagenesis to understand enzyme-substrate interactions.

Main Results:

• Identified five P450 enzymes with activity against alkaloid scaffolds.
• Discovered four enzymes from distinct plant species that selectively oxygenated the anticancer scaffold evodiamine at two positions.
• Elucidated the role of active site hydrophobic bulk in governing substrate positioning and catalytic selectivity.

Conclusions:

• Orthology-guided enzyme discovery combined with biocatalysis enables selective functionalization of pharmacologically active alkaloids.
• This integrated approach facilitates the structural diversification of complex natural products for drug development.
• The findings open new avenues for engineering enzymes for targeted modification of valuable small molecules.