Substrate-Triggered Formation and Remarkable Stability of the C−H Bond-Cleaving Chloroferryl Intermediate in the Aliphatic Halogenase, SyrB2

Aliphatic halogenases activate O2, cleave α-ketoglutarate (αKG) to CO2 and succinate, and form haloferryl [X−Fe(IV)═O; X = Cl or Br] complexes that cleave aliphatic C−H bonds to install halogens during the biosynthesis of natural products by non-ribosomal peptide synthetases (NRPSs). For the related αKG-dependent dioxygenases, it has been shown that reaction of the Fe(II) cofactor with O2 to form the C−H bond-cleaving ferryl complex is “triggered” by binding of the target substrate. In this study, we have tested for and defined structural determinants of substrate triggering (ST) in the halogenase, SyrB2, from the syringomycin E biosynthetic NRPS of Pseudomonas syringae B301D. As for other halogenases, the substrate of SyrB2 is complex, consisting of l-Thr tethered via a thioester linkage to a covalently bound phosphopantetheine (PPant) cofactor of a carrier protein, SyrB1. Without an appended amino acid, SyrB1 does not trigger formation of the chloroferryl intermediate state in SyrB2, even in the presenc...