Functional Characterization and Molecular Basis of a Multi‐site Halogenase in Naphthacemycin Biosynthesis

Halogenases are spurring a growing interest in the fields of biosynthesis and biocatalysis. Despite various halogenases have been identified in numerous natural product biosynthetic pathways, the mechanisms for multiple halogenations and site‐selectivity remain largely unclear. In this study, we biochemically characterize FasV, a FAD‐dependent halogenase (FDH) that catalyzes five successive chlorinations in the biosynthesis of naphthacene‐containing aromatic polyketide naphthacemycin. This multiple halogenation reaction is elucidated occurring in an orderly fashion, as evidenced by enzyme kinetics, time‐course assays, and computational simulations. Crystallographic analyses and mutagenesis studies reveal previously unrecognized amino acid residues, including T53, L81, F93, and I212, that are crucial for controlling regioselectivity and substrate specificity. Based on this, a I212T mutant is generated to exclusively catalyze selective monohalogenation. We propose a novel dual‐activation mechanism, and demonstrate that FasV’s larger binding pocket makes it a valuable biocatalyst for other substrate with diverse structure. Therefore, this study provides new insights into multi‐site polyhalogenases and highlights the potential for engineering FasV‐like FDHs for biocatalytic applications.