The founding members of the HD-domain protein superfamily are phosphohydrolases, and newly discovered members are generally annotated as such. However, myo-inositol oxygenase (MIOX) exemplifies a second, very different function that has evolved within the common scaffold of this superfamily. A recently discovered HD protein, PhnZ, catalyzes conversion of 2-amino-1-hydroxyethylphosphonate to glycine and phosphate, culminating a bacterial pathway for the utilization of environmentally abundant 2-aminoethylphosphonate. Using Mössbauer and EPR spectroscopies, X-ray crystallography, and activity measurements, we show here that, like MIOX, PhnZ employs a mixed-valent Fe(II)/Fe(III) cofactor for the O2-dependent oxidative cleavage of its substrate. Phylogenetic analysis suggests that many more HD proteins may catalyze yet-unknown oxygenation reactions using this hitherto exceptional Fe(II)/Fe(III) cofactor. The results demonstrate that the catalytic repertoire of the HD superfamily extends well beyond phosphohydrolysis and suggest that the mechanism used by MIOX and PhnZ may be a common strategy for oxidative C-X bond cleavage.
Three-dimensional structures of PhnZ shown in comparison with MIOX. (A) Ribbon diagram illustrating the α-helical fold of PhnZ, the location of the diiron site, and coordination by a citrate molecule. A loop region (residues 66–72) that could not be modeled is represented as a dotted line. An Fe anomalous difference Fourier map (orange mesh, contoured at 7.0 σ) is shown in Inset. Also shown are views of the active site in PhnZ with the citrate ligand (B), MIOX with the substrate myo-inositol (C), and PhnZ with the substrate (R)-OH-AEP (D). Amino acid side chains in the first coordination sphere and substrate molecules are represented in stick format, FeIII ions are shown as orange spheres, and nonprotein oxygen-based ligands are shown as red spheres. Because of the modest resolution (3.0 Å) of the (R)-OH-AEP–bound PhnZ structure, the oxo/hydoxo bridge cannot be resolved and, therefore, was not modeled.