Structure of a Novel Phosphotriesterase from Sphingobium sp. TCM1: A Familiar Binuclear Metal Center Embedded in a Seven-Bladed β-Propeller Protein Fold.

Abstract

A novel phosphotriesterase was recently discovered and purified from Sphingobium sp. TCM1 (Sb-PTE) and shown to catalyze the hydrolysis of a broad spectrum of organophosphate esters with a catalytic efficiency that exceeds 10(6) M(-1) s(-1) for the hydrolysis of triphenyl phosphate. The enzyme was crystallized and the three-dimensional structure determined to a resolution of 2.1 Å using single-wavelength anomalous diffraction (Protein Data Bank entry 5HRM ). The enzyme adopts a seven-bladed β-propeller protein fold, and three disulfide bonds were identified between Cys-146 and Cys-242, Cys-411 and Cys-443, and Cys-542 and Cys-559. The active site of Sb-PTE contains a binuclear manganese center that is nearly identical to that of the structurally unrelated phosphotriesterase from Pseudomonas diminuta (Pd-PTE). The two metal ions in the active site are bridged to one another by Glu-201 and a water molecule. The α-metal ion is further coordinated to the protein by interactions with His-389, His-475, and Glu-407, whereas the β-metal ion is further liganded to His-317 and His-258. Computational docking of mimics of the proposed pentavalent reaction intermediates for the hydrolysis of organophosphates was used to provide a model for the binding of chiral substrates in the active site of Sb-PTE. The most striking difference in the catalytic properties of Sb-PTE, relative to those of Pd-PTE, is the enhanced rate of hydrolysis of organophosphate esters with substantially weaker leaving groups. The structural basis for this difference in the catalytic properties between Sb-PTE and Pd-PTE, despite the nearly identical binuclear metal centers for the activation of the substrate and nucleophilic water molecule, is at present unclear.