A second-sphere threonine residue directs iron-oxygen species in a Fe(II)/α-ketoglutarate-dependent isonitrile synthase

Threonine T92 in the Fe(II)/α-ketoglutarate-dependent dioxygenase Rv0097 directs iron–oxygen species by coordinating online H-bonding, KG positioning, and loop stabilization during isonitrile synthesis.

• Why it matters: Isonitrile biosynthesis by Rv0097 requires two half-reactions using a highly reactive Fe(IV)-oxo intermediate, but how second-sphere residues tune iron-oxygen reactivity and co-substrate access was unclear.
• What they did: The study compared atomic-resolution crystal structures with biochemical and computational data to identify T92’s roles in hydrogen bonding to O2 and Fe(IV)-oxo, offline KG binding, and post–first half-reaction loop stabilization.
• The result: T92 is linked to controlling HAT from the substrate carbon and enabling KG cleavage products to leave and a second KG to bind, suggesting analogous second-sphere residues in other Fe(II)/KG dioxygenases for rational engineering.

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