Crystal structure of PhnZ in complex with substrate reveals a di-iron oxygenase mechanism for catabolism of organophosphonates

Abstract

The enzymes PhnY and PhnZ comprise an oxidative catabolic pathway that enables marine bacteria to use 2-aminoethylphosphonic acid as a source of inorganic phosphate. PhnZ is notable for catalyzing the oxidative cleavage of a carbon-phosphorus bond using Fe(II) and dioxygen, despite belonging to a large family of hydrolytic enzymes, the HD-phosphohydrolase superfamily. We have determined high-resolution structures of PhnZ bound to its substrate, (R)-2-amino-1-hydroxyethylphosphonate (2.1 Å), and a buffer additive, l-tartrate (1.7 Å). The structures reveal PhnZ to have an active site containing two Fe ions coordinated by four histidines and two aspartates that is strikingly similar to the carbon-carbon bond cleaving enzyme, myo-inositol-oxygenase. The exception is Y24, which forms a transient ligand interaction at the dioxygen binding site of Fe2. Site-directed mutagenesis and kinetic analysis with substrate analogs revealed the roles of key active site residues. A fifth histidine that is conserved in the PhnZ subclade, H62, specifically interacts with the substrate 1-hydroxyl. The structures also revealed that Y24 and E27 mediate a unique induced-fit mechanism whereby E27 specifically recognizes the 2-amino group of the bound substrate and toggles the release of Y24 from the active site, thereby creating space for molecular oxygen to bind to Fe2. Structural comparisons of PhnZ reveal an evolutionary connection between Fe(II)-dependent hydrolysis of phosphate esters and oxidative carbon-phosphorus or carbon-carbon bond cleavage, thus uniting the diverse chemistries that are found in the HD superfamily.

Keywords: C–H bond activation; C–P bond cleavage; nonheme iron-dependent oxygenase; phosphonate.

Fig. 1.

Oxidative conversion of 2-aminoethylphosphonic acid (1) to glycine (3) and Pi (4) by PhnY and PhnZ. Compounds 5 and 6 are substrate analogs used in this study.

Fig. 2.

Overall structure of PhnZ. (A) The structure of PhnZ, molecule A, bound to l-tartrate. The two Fe ions are shown as orange spheres, l-tartrate as green sticks. (Inset) 2Fo-Fc omit map observed for l-tartrate contoured to 2σ (blue) and the 2Fo-Fc omit map for the Fe ions contoured to 5σ (pink). (B) Cartoon representation of the structure of PhnZ, molecule A, bound to the substrate (R)-2 (shown as yellow and orange sticks), and overlaid with the surface representation of the l-tartrate–bound structure (gray). (Inset) 2Fo-Fc omit map observed for (R)-2 contoured to 2σ (blue) and the 2Fo-Fc omit map for the Fe ions contoured to 5σ (magenta). Major conformational differences between the two structures involving a large movement of the H62-H80 loop (pink) and the D21-N30 loop (teal) are observed at the mouth of the active site.

Fig. 3.

Substrate and Fe ion coordination in the PhnZ active site. (A) PhnZ, molecule A, bound to l-tartrate, shown in green sticks.(B) PhnZ, molecule A, bound to (R)-2, shown in yellow and orange sticks. (C) PhnZ, molecule B, bound to (R)-2. Disordered residues are represented by dashed lines. In all figure parts the H62-H80 loop is colored pink and the D21-N30 loop is teal. Fe ions and water molecules are shown as orange and red spheres, respectively. Corresponding active site schemes are shown with interatomic distances indicated in Å (Lower). Stereo views of the active sites can be found in SI Appendix, Fig. S2.

Fig. 4.

PhnZ is structurally homologous to Fe(II)-dependent enzymes of the HD hydrolase superfamily. (A) Active site of PhnZ, molecule A, bound to the substrate (R)-2. The substrate is shown in yellow and orange sticks, Fe ions as orange spheres, and water or hydroxide ions as red spheres. (B) Active site of MIOX (PDB ID code 2huo). The substrate myo-inositol is shown as gray sticks. (C) Active site of Streptococcus agalactiae phosphohydrolase (PDB ID code 2ogi) bound to GDP (shown as gray and orange sticks). (D) Hypothetical mechanism for an Fe(II)-dependent phosphohydrolase.

Fig. 5.

Proposed mechanism for PhnZ, highlighting the roles of Y24, E27, and H62.