Unlocking the catalytic precision of ligand-controlled enzymatic halogenation
Aisaraphon Phintha1, April L Lukowski2,3, Pimchai Chaiyen1
1School of Biomolecular Science and Engineering, Vidyasirimedhi Institute of Science and Technology (VISTEC), Rayong 10120, Thailand.
This study reveals how the unique flavin-dependent halogenase AetF controls its catalytic cycle. Ligand binding, specifically NADP+ and L-tryptophan, is crucial for activating halogenation and accelerating the reaction.
Area of Science:
- Biochemistry
- Enzymology
- Biocatalysis
Background:
- Flavin-dependent halogenases are valuable biocatalysts.
- The enzyme AetF's unique structure complicates prediction of its flavin chemistry.
Purpose of the Study:
- To investigate the flavin reaction mechanisms of AetF.
- To understand the role of substrate binding in controlling AetF's catalytic activity.
Main Methods:
- Transient kinetics studies to analyze flavin intermediates.
- Structural and tunnel analyses to elucidate ligand-induced conformational changes.
- Light-induced flavin reduction and NADP+ stimulation for biocatalytic applications.
Main Results:
- NADP+ binding is essential for forming the C4a-hydroperoxy flavin adenine dinucleotide (FAD) intermediate.
- Flavin intermediates are stabilized without L-tryptophan but rapidly dehydrate in its presence.
- Substrate binding significantly accelerates the catalytic cycle of AetF.
Conclusions:
- AetF precisely controls its flavin chemistry through ligand binding.
- Protein tunnel alterations induced by NADP+ and L-tryptophan binding are key to this control.
- Mechanistic insights can guide the biocatalytic development of halogenases.
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