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bioRxiv · 10.1101/2025.10.24.684477

Separation of halide oxidation and substrate halogenation chemistries rationalizes site-selective vanadium dependent haloperoxidase catalysis

Baumgartner, J. T.·Varga, L. A.·Calhoun, J. T.·Serrao, V. H. B.·Loerch, S.·McKinnie, S. M. K.
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Abstract

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 Enhygromyxa salina (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 support for an intermediate bromamine involved in selectivity. Cryo-EM of the BrOx state reveals a camera shutter mechanism that compacts the halide entry tunnel and vanadate pocket, minimizing the premature dissociation of hypohalous acid. These findings collectively unveil a multilayered halogen trapping and transfer mechanism and provide a rationale for selective VHPO catalysis. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/684477v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@6154b9org.highwire.dtl.DTLVardef@169fdorg.highwire.dtl.DTLVardef@d56940org.highwire.dtl.DTLVardef@f11028_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Baumgartner, J. T., Varga, L. A., Calhoun, J. T., Serrao, V. H. B., Loerch, S., McKinnie, S. M. K.. 2025-10-25. Separation of halide oxidation and substrate halogenation chemistries rationalizes site-selective vanadium dependent haloperoxidase catalysis. https://doi.org/10.1101/2025.10.24.684477

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