Substrate-Triggered μ-Peroxodiiron(III) Intermediate in the 4-Chloro-L-Lysine-Fragmenting Heme-Oxygenase-like Diiron Oxidase (HDO) BesC: Substrate Dissociation from, and C4 Targeting by, the Intermediate

The enzyme BesC from the beta-ethynyl-L-serine biosynthetic pathway in Streptomyces cattleya fragments 4-chloro-L-lysine (produced from L-Lysine by BesD) to ammonia, formaldehyde, and 4-chloro-L-allylglycine and can analogously fragment L-Lys itself. BesC belongs to the emerging family of O-2 activating non-heme-diiron enzymes with the heme-oxygenaselike protein fold (HDOs). Here, we show that the binding of L-Lys or an analogue triggers capture of O-2 by the protein's diiron(II) cofactor to form a blue p-peroxodiiron(III) intermediate analogous to those previously characterized in two other HDOs, the olefininstalling fatty acid decarboxylase, UndA, and the guanidino-Noxygenase domain of SznF. The similar to 5- and similar to 30-fold faster decay of the intermediate in reactions with 4-thia-L-Lys and (4RS)-chloroDL-lysine than in the reaction with L-Lys itself and the primary deuterium kinetic isotope effects (D-KIEs) on decay of the intermediate and production of L-allylglycine in the reaction with 4,4,5,5-[H-2(4)]-L-Lys suggest that the peroxide intermediate or a reversibly connected successor complex abstracts a hydrogen atom from C4 to enable olefin formation. Surprisingly, the sluggish substrate L-Lys can dissociate after triggering intermediate formation, thereby allowing one of the better substrates to bind and react. The structure of apo BesC and the demonstrated linkage between Fe(II) and substrate binding suggest that the triggering event involves an induced ordering of ligand-providing helix 3 (alpha 3) of the conditionally stable HDO core. As previously suggested for SznF, the dynamic alpha 3 also likely initiates the spontaneous degradation of the diiron(III) product cluster after decay of the peroxide intermediate, a trait emerging as characteristic of the nascent HDO family.

Automated summary

A newly discovered enzyme BesC from Streptomyces cattleya has been found to catalyze the biosynthesis of beta-ethynyl-L-serine. The enzyme can fragment 4-chloro-L-lysine and L-lysine itself, and the degradation of the intermediate formed during the reaction is influenced by the presence of different analogues. The study also suggests that hydrogen atom extraction plays a role in olefin formation.