@article{ad785d66d76b424f8f1370a86a257ee6,
title = "Structural Basis of Stereospecific Vanadium-Dependent Haloperoxidase Family Enzymes in Napyradiomycin Biosynthesis",
abstract = "Vanadium-dependent haloperoxidases (VHPOs) from Streptomyces bacteria differ from their counterparts in fungi, macroalgae, and other bacteria by catalyzing organohalogenating reactions with strict regiochemical and stereochemical control. While this group of enzymes collectively uses hydrogen peroxide to oxidize halides for incorporation into electron-rich organic molecules, the mechanism for the controlled transfer of highly reactive chloronium ions in the biosynthesis of napyradiomycin and merochlorin antibiotics sets the Streptomyces vanadium-dependent chloroperoxidases apart. Here we report high-resolution crystal structures of two homologous VHPO family members associated with napyradiomycin biosynthesis, NapH1 and NapH3, that catalyze distinctive chemical reactions in the construction of meroterpenoid natural products. The structures, combined with site-directed mutagenesis and intact protein mass spectrometry studies, afforded a mechanistic model for the asymmetric alkene and arene chlorination reactions catalyzed by NapH1 and the isomerase activity catalyzed by NapH3. A key lysine residue in NapH1 situated between the coordinated vanadate and the putative substrate binding pocket was shown to be essential for catalysis. This observation suggested the involvement of the ϵ-NH2, possibly through formation of a transient chloramine, as the chlorinating species much as proposed in structurally distinct flavin-dependent halogenases. Unexpectedly, NapH3 is modified post-translationally by phosphorylation of an active site His (τ-pHis) consistent with its repurposed halogenation-independent, α-hydroxyketone isomerase activity. These structural studies deepen our understanding of the mechanistic underpinnings of VHPO enzymes and their evolution as enantioselective biocatalysts.",
author = "Chen, {Percival Yang Ting} and Sanjoy Adak and Chekan, {Jonathan R.} and Liscombe, {David K.} and Akimasa Miyanaga and Peter Bernhardt and Stefan Diethelm and Fielding, {Elisha N.} and George, {Jonathan H.} and Miles, {Zachary D.} and Murray, {Lauren A.M.} and Steele, {Taylor S.} and Winter, {Jaclyn M.} and Noel, {Joseph P.} and Moore, {Bradley S.}",
note = "Funding Information: This research was supported by the National Institutes of Health via Grant R01-AI047818 to B.S.M. and Grant F32-GM096711 to P.B., the Howard Hughes Medical Institute and the National Science Foundation via Grant EEC-0813570 to J.P.N., and fellowships from the Life Science Research Foundation through a Simons Foundation Fellowship (J.R.C.), the Natural Sciences and Engineering Research Council of Canada (D.K.L.), the Japan Society for the Promotion of Science (21-644, A.M.), the Swiss National Science Foundation (S.D.), and the National Science Foundation (T.S.S.). Funding Information: The authors thank G. Louie (The Salk Institute for Biological Studies) and the staff of the Advanced Light Source (ALS) at beamlines 8.2.1 and 8.2.2 for assistance with data collection and Y. Su and staff at the Molecular Mass Spectrometry Facility (MMSF, University of California, San Diego) for the mass spectroscopy measurements. The Agilent 6230 ESI-TOFMS instrument at MMSF was supported by National Institutes of Health (NIH) Grant 1S10RR25636-1A1, and the ALS beamlines are a U.S. Department of Energy Office of Science User Facility under Contract DE-AC02-05CH11231 and supported in part by the ALS-ENABLE program funded by NIH grant P30 GM124169-01. Funding Information: The authors thank G. Louie (The Salk Institute for Biological Studies) and the staff of the Advanced Light Source (ALS) at beamlines 8.2.1 and 8.2.2 for assistance with data collection and Y. Su and staff at the Molecular Mass Spectrometry Facility (MMSF University of California, San Diego) for the mass spectroscopy measurements. The Agilent 6230 ESI-TOFMS instrument at MMSF was supported by National Institutes of Health (NIH) Grant 1S10RR25636-1A1, and the ALS beamlines are a U.S. Department of Energy Office of Science User Facility under Contract DE-AC02-05CH11231 and supported in part by the ALS-ENABLE program funded by NIH grant P30 GM124169-01. Publisher Copyright: {\textcopyright} 2022 American Chemical Society. All rights reserved.",
year = "2022",
month = sep,
day = "6",
doi = "10.1021/acs.biochem.2c00338",
language = "English",
volume = "61",
pages = "1844--1852",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "17",
}