Structural and functional insights into the self-sufficient flavin-dependent halogenase

Area of Science:

• Biochemistry
• Synthetic Chemistry
• Structural Biology

Background:

• Flavin-dependent halogenases (FDHs) are versatile enzymes with significant applications in synthetic chemistry.
• Single-component FDHs, like AetF, possess both halogenase and reductase activities within one polypeptide chain.
• AetF demonstrates broad substrate promiscuity, catalyzing the sequential bromination of l-tryptophan (l-Trp) to 5-bromotryptophan (5-Br-Trp) and 5,7-dibromo-l-tryptophan (5,7-di-Br-Trp).

Purpose of the Study:

• To elucidate the catalytic mechanism of the single-component flavin-dependent halogenase AetF.
• To determine the crystal structure of AetF in complex with its cofactors and substrate intermediates.
• To understand the structural basis for AetF's substrate flexibility and dibromination capability.

Main Methods:

• X-ray crystallography was employed to solve the crystal structure of AetF.
• Structures were determined in complex with FAD, FAD/NADP+, FAD/l-Trp, and FAD/5-Br-Trp.
• Structural analysis focused on substrate-binding pocket topology and residue interactions.

Main Results:

• The crystal structures revealed an unprecedented topology for a single-component FDH.
• A spacious substrate-binding pocket in AetF contributes to its substrate flexibility and dibromination capacity.
• Specific interaction networks between substrate-recognizing residues and 5-Br-Trp are critical for dibromination.
• Engineered Ala variants of AetF achieved >98% C5-regioselectivity for l-Trp monobromination.

Conclusions:

• The study provides the first detailed mechanistic insights into single-component FDH activity.
• The findings offer a structural basis for understanding AetF's catalytic mechanism and regioselectivity.
• This work facilitates protein engineering efforts for developing efficient FDHs in biocatalytic halogenation.