Synergistic Binding of the Halide and Cationic Prime Substrate of l-Lysine 4-Chlorinase, BesD, in Both Ferrous and Ferryl States

Jeffrey W. Slater, Chi Yun Lin, Monica E. Neugebauer, Molly J. McBride, Debangsu Sil, Mrutyunjay A. Nair, Bryce J. Katch, Amie K. Boal, Michelle C.Y. Chang, Alexey Silakov, Carsten Krebs, J. Martin Bollinger

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Abstract

An aliphatic halogenase requires four substrates: 2-oxoglutarate (2OG), halide (Cl- or Br-), the halogenation target (“prime substrate”), and dioxygen. In well-studied cases, the three nongaseous substrates must bind to activate the enzyme’s Fe(II) cofactor for efficient capture of O2. Halide, 2OG, and (lastly) O2 all coordinate directly to the cofactor to initiate its conversion to a cis-halo-oxo-iron(IV) (haloferryl) complex, which abstracts hydrogen (H) from the non-coordinating prime substrate to enable radicaloid carbon-halogen coupling. We dissected the kinetic pathway and thermodynamic linkage in binding of the first three substrates of the l-lysine 4-chlorinase, BesD. After addition of 2OG, subsequent coordination of the halide to the cofactor and binding of cationic l-Lys near the cofactor are associated with strong heterotropic cooperativity. Progression to the haloferryl intermediate upon the addition of O2 does not trap the substrates in the active site and, in fact, markedly diminishes cooperativity between halide and l-Lys. The surprising lability of the BesD•[Fe(IV)=O]•Cl•succinate•l-Lys complex engenders pathways for decay of the haloferryl intermediate that do not result in l-Lys chlorination, especially at low chloride concentrations; one identified pathway involves oxidation of glycerol. The mechanistic data imply (i) that BesD may have evolved from a hydroxylase ancestor either relatively recently or under weak selective pressure for efficient chlorination and (ii) that acquisition of its activity may have involved the emergence of linkage between l-Lys binding and chloride coordination following the loss of the anionic protein-carboxylate iron ligand present in extant hydroxylases.

Original languageEnglish (US)
Pages (from-to)2480-2491
Number of pages12
JournalBiochemistry
Volume62
Issue number16
DOIs
StatePublished - Aug 15 2023

All Science Journal Classification (ASJC) codes

  • Biochemistry

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