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

1. Laura M. van Staalduinena, 
2. Fern R. McSorleyb, 
3. Katharina Schiesslc, 
4. Jacqueline Séguinb, 
5. Peter B. Wyattd,
6. Friedrich Hammerschmidtc, 
7. David L. Zechelb,1, and 
8. Zongchao Jiaa,1

Significance

Inorganic phosphate (Pi) is an essential component of many biological molecules and thus is required by all life forms. However, soluble Pi is typically at low abundance in the environment. To compensate, microbes have evolved unique carbon–phosphorus-bond cleaving reactions to use organophosphonates as an alternative source of Pi. The marine-derived enzyme PhnZ utilizes a new oxidative mechanism for CP bond cleavage involving iron and molecular oxygen. The three-dimensional structure of PhnZ reveals unique active site features that contribute to catalysis of CP bond cleavage and substrate specificity, as well as an evolutionary link between phosphodiester bond hydrolysis and oxidative bond cleavage. This evolutionary link likely reflects the ancient origins of organophosphonates in the environment.

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.

• phosphonate
 
• nonheme iron-dependent oxygenase
 
• C–P bond cleavage
 
• C–H bond activation