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

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 non- gaseous substrates must bind to activate the enzyme's Fe(II) cofactor for efficient capture of O2. Halide, 2OG, and (lastly) O2all 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 2OG adds, 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 addition of O2does 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 that (i) 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 loss of the anionic protein-carboxylate iron ligand present in extant hydroxylases.

An Unusual Ferryl Intermediate and Its Implications for the Mechanism of Oxacyclization by the Loline-Producing Iron(II)- and 2-Oxoglutarate-Dependent Oxygenase, LolO

“…The results imply that LolO preferentially cyclizes 2-OH-AcAP but, in competition, also hydroxylates it to a minor extent (at C7; see below), as has been seen previously with other Fe/2OG enzymes (e.g., halogenases and H6H). 41,50,51 When LolO cyclization reactions were conducted under 18 O 2 , the Δm/z of the primary NANL product was not affected, as expected for the coupling of the extant C2−O to C7 in the formation of the cyclic ether (Figure 1A). However, the minor product, previously at Δm/z +16, was shifted by two mass units to Δm/z+18 (inset of Figure 1A), implying that it results from an aberrant second hydroxylation by oxygen rebound.…”

“…However, the minor product, previously at Δm/z +16, was shifted by two mass units to Δm/z+18 (inset of Figure 1A), implying that it results from an aberrant second hydroxylation by oxygen rebound. The incorporation of 18 O is discussed more quantitatively below. S2).…”