Fig. 2.

HygX structure. (A) HygX with major strands in yellow and minor strands in red. Ni²⁺and Fe²⁺ are green and orange spheres. (B) HygX A chain colored by crystallographic temperature factors where cool colors indicate low B-factors. Loop insertions are highlighted. (C) Surface representation in the same orientation as B. (D) Superposition of four chains of the HygX-AKG complex showing loop movement, with hygromycin B shown for reference. (E) Iron coordination with anomalous difference density in yellow and contoured at 5σ. (F) Active site metal coordination of HygX. |F_O| − |F_C| difference density is contoured around AKG at 2.5 σ, calculated before inclusion of AKG in the model. (G) Tryptophan fluorescence at 350 nm of 0.5 μM HygX, 0.05 mM NiCl₂, 0.05 mM AKG, and varying concentrations of hygromycin B (K_d, 3.4 ± 0.5 μM). (H) 3σ |F_O| − |F_C| difference density calculated before hygromycin B placement. One possible site of hydrogen atom abstraction is 5.2 Å from the metal center. (I) HygX–AKG–hygromycin B product complex (in gray) aligned with the clavaminate synthase–AKG–proclavaminate substrate complex (blue; PDB ID code 1DRT). Asterisks mark sites of hydrogen atom abstraction.