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

An aliphatic halogenase requires four substrates: 2-oxoglutarate(2OG), halide (Cl- or Br-), thehalogenation target (prime substrate), and dioxygen.In well-studied cases, the three nongaseous substrates must bind toactivate the enzyme's Fe(II) cofactor for efficient captureof O-2. Halide, 2OG, and (lastly) O-2 all coordinatedirectly to the cofactor to initiate its conversion to a cis-halo-oxo-iron(IV) (haloferryl) complex, which abstracts hydrogen(H-& BULL;) from the non-coordinating prime substrate toenable radicaloid carbon-halogen coupling. We dissected thekinetic pathway and thermodynamic linkage in binding of the firstthree substrates of the l-lysine 4-chlorinase, BesD. Afteraddition of 2OG, subsequent coordination of the halide to the cofactorand binding of cationic l-Lys near the cofactor are associatedwith strong heterotropic cooperativity. Progression to the haloferrylintermediate upon the addition of O-2 does not trap thesubstrates in the active site and, in fact, markedly diminishes cooperativitybetween halide and l-Lys. The surprising lability of theBesD & BULL;[Fe(IV)=O]& BULL;Cl & BULL;succinate & BULL;l-Lys complex engenders pathways for decay of the haloferryl intermediatethat do not result in l-Lys chlorination, especially at lowchloride concentrations; one identified pathway involves oxidationof glycerol. The mechanistic data imply (i) that BesD may have evolvedfrom a hydroxylase ancestor either relatively recently or under weakselective pressure for efficient chlorination and (ii) that acquisitionof its activity may have involved the emergence of linkage between l-Lys binding and chloride coordination following the loss ofthe anionic protein-carboxylate iron ligand present in extant hydroxylases.

Automated summary

An aliphatic halogenase requires four substrates: 2-oxoglutarate(2OG), halide (Cl- or Br-), the halogenation target (prime substrate), and dioxygen. The binding of 2OG, halide, and l-Lys to the cofactor of the enzyme activates the Fe(II) for O-2 capture and initiates its conversion to a haloferryl complex. The mechanistic data of BesD suggest that it may have evolved from a hydroxylase ancestor and the acquisition of its activity involves a linkage between l-Lys binding and chloride coordination.