SyrB2 halogenase inserts halogens (Cl or Br) into the unactivated C-H bond of its substrate threonine using a high-valent haloferryl intermediate (X-Fe<sup>IV</sup>=O) with high chemoselectivity under mild, aqueous conditions. When threonine is substituted for non-native amino acid substrates, the chemoselectivity shifts to favor hydroxylated products over halogenated ones. While it’s well-understood how SyrB2 leverages substrate-protein interactions to influence chemoselectivity, it’s unknown how the protein interacts with the haloferryl ligands to promote chlorination activity. In this ACS presentation, I will discuss our studies towards understanding how protein-guided secondary coordination sphere (SCS) interactions influence reaction outcome. We hypothesize that SyrB2’s reaction outcome may be determined by the identity of the haloferryl isomer and that SyrB2 leverages SCS interactions to align a specific isomer that will preferentially chlorinate substrates. To test this hypothesis, we computationally designed several SyrB2 variants that interact with the oxo ligand of a specific haloferryl isomer. After performing halogenation assays, we assessed the chemo-selectivity of each variant with a range of amino acid substrates. Our results will validate the identity of the haloferryl intermediate in SyrB2 and inform future biocatalytic halogenation strategies.

Catalytic influences of the secondary coordination sphere in an iron halogenase

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ACS Fall 2022

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