biostudies-literatureUnknownBaumgartner JTNIGMS NIH HHSVanadium-dependent haloperoxidases (VHPOs) catalyze the halogenation of organic molecules under mild aqueous conditions. Selective bacterial VHPOs exhibit exquisite regio- and enantiocontrol, however the precise mechanisms dictating selectivity have remained elusive. We have solved the single-particle cryo-electron microscopy (cryo-EM) structure of a selective bromoperoxidase from <i>Enhygromyxa salina</i> (esVHPO). Mutagenesis demonstrates that halide oxidation and substrate halogenation occur in two distinct pockets, with halide transfer mediated by critical lysine residue K329. Isolation of a stable intermediate following bromide oxidation (BrOx) enables single turnover catalysis in the presence of organic substrate; subsequent application of a chemoselective fluorescent probe provides bioRxiv : the preprint server for biology2025.10.24.684477https://www.ebi.ac.uk/biostudies/studies/S-EPMC12633397biostudies-literatureSeparation of halide oxidation and substrate halogenation chemistries rationalizes site-selective vanadium dependent haloperoxidase catalysis.PMC12633397R01 GM129325R35 GM147235U24 GM129547R24 GM154185McKinnie SMKBaumgartner JTLoerch SVarga LACalhoun JTSerrao VHBfalseSeparation of halide oxidation and substrate halogenation chemistries rationalizes site-selective vanadium dependent haloperoxidase catalysis.Vanadium-dependent haloperoxidases (VHPOs) catalyze the halogenation of organic molecules under mild aqueous conditions. Selective bacterial VHPOs exhibit exquisite regio- and enantiocontrol, however the precise mechanisms dictating selectivity have remained elusive. We have solved the single-particle cryo-electron microscopy (cryo-EM) structure of a selective bromoperoxidase from <i>Enhygromyxa salina</i> (esVHPO). Mutagenesis demonstrates that halide oxidation and substrate halogenation occur in two distinct pockets, with halide transfer mediated by critical lysine residue K329. Isolation of a stable intermediate following bromide oxidation (BrOx) enables single turnover catalysis in the presence of organic substrate; subsequent application of a chemoselective fluorescent probe provides 2025-01-01T00:00:00Z2025 Oct2026-06-04T03:19:07.85Z2026-06-04T03:12:25.689ZS-EPMC126333974128011610.1101/2025.10.24.684477