Discovery and Characterization of a Single-Component Halogenase for Phenazine Halogenation.

Halogenated phenazines hold promise as antimicrobial and antibiofilm agents, yet are mainly accessed via chemical synthesis. Herein, we report PezW, a novel single-component flavin-dependent halogenase (FDH) that halogenates phenazine scaffolds, notably enabling enzymatic synthesis of bioactive 2-bromo-1-hydroxyphenazine () and 2,4-bromo-1-hydroxyphenazine (). Structural modeling and mutagenesis revealed key residues critical for substrate binding and catalysis. These findings highlight PezW's potential as a biocatalyst for the synthesis of halogenated phenazines with pharmaceutical relevance.