Site-Selective C-H Halogenation Using Flavin-Dependent Halogenases Identified via Family-Wide Activity Profiling

Area of Science:

• Biocatalysis
• Enzyme Engineering
• Organic Chemistry

Background:

• Enzyme catalysis is crucial for selective C-H bond functionalization.
• Predicting enzyme activity from sequence remains a significant challenge in biocatalysis.
• Flavin-dependent halogenases (FDHs) are a class of enzymes with potential for C-H functionalization.

Purpose of the Study:

• To conduct family-wide activity profiling of flavin-dependent halogenases (FDHs).
• To gain insights into FDH sequence-function relationships for improved biocatalyst identification.
• To identify novel FDHs for efficient late-stage C-H functionalization of complex molecules.

Main Methods:

• Employed family-wide activity profiling to assess FDH enzyme variants.
• Characterized novel FDHs for substrate scope and regioselectivity.
• Utilized identified FDHs for preparative-scale late-stage C-H functionalization.

Main Results:

• Generated comprehensive sequence-function data for FDHs, revealing halide specificity and substrate preferences.
• Identified specific regions within FDH sequence space likely to yield enzymes for small-molecule halogenation.
• Discovered FDHs with novel substrate scope and complementary regioselectivity for complex compounds.

Conclusions:

• Family-wide profiling offers significant time savings for biocatalyst discovery compared to directed evolution.
• The identified FDHs serve as advanced starting points for further enzyme evolution and application.
• This strategy accelerates the development of enzymes for challenging C-H functionalization reactions.