Iron(II) and 2-oxoglutarate-dependent oxygenases represent a diverse family of enzymes capable of harnessing molecular oxygen to functionalize chemically inert C-H bonds. Though the majority of these enzymes hydroxylate their substrate, a variety of other reaction outcomes including halogenation, stereoinversion, and desaturation have been observed. Of these reactions, halogenation is a transformation of special interest to the pharmaceutical and agrochemical industries as halogens play a central role in modulating the bioactivity of small molecules. Halogenation is also a valuable transformation to the synthetic chemist, as halogen reactivity enables selective functionalization in even late stages of a synthesis.<br/><br/>A recently discovered Fe(II)/2OG halogenase, BesD, shows particular promise as a biocatalyst. Previously characterized Fe(II)/2OG halogenases required either large carrier proteins or highly complex substrates, making synthetic applications and mechanistic studies difficult. In contrast, BesD accepts the canonical amino acid L-lysine, independent of a carrier protein. Beyond its native substrate, BesD has also been shown to chlorinate the urea cycle metabolite L-ornithine and lysine isomer D-lysine in moderate amounts.<br/><br/>The previously demonstrated reactivity of this halogenase opens the door for further mechanistic investigation. In this work, we examine the kinetics underpinning the reactivity of BesD with L-lysine and various substrate analogs. We also investigate the dependence of substrate binding and reactive intermediate formation on the anion identity and concentration. This work sheds light on how Fe(II)/2OG enzymes can perform halogenation in contrast to the well-characterized family of 2OG-hydroxylases. Furthermore, with a greater mechanistic understanding of 2OG-dependent halogenation, the reaction conditions can be optimized to afford a synthetically useful catalyst.

Mechanistic investigation of the iron(II) and 2-oxoglutarate-dependent halogenase, BesD

ACS Spring 2022

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