Substrate Profile of the Phosphotriesterase Homology Protein from Escherichia coli

Abstract

The phosphotriesterase homology protein (PHP) from Escherichia coli is a member of a family of proteins that is related to phosphotriestrase (PTE), a bacterial enzyme from cog1735 with unusual substrate specificity toward the hydrolysis of synthetic organic phosphates and phosphonates. PHP was cloned, purified to homogeneity, and functionally characterized. The three-dimensional structure of PHP was determined at a resolution of 1.84 Å with zinc and phosphate in the active site. The protein folds as a distorted (β/α)₈-barrel and possesses a binuclear metal center in the active site. The catalytic function and substrate profile of PHP were investigated using a structure-guided approach that combined bioinformatics, computational docking, organic synthesis, and steady-state enzyme kinetics. PHP was found to catalyze the hydrolysis of phosphorylated glyceryl acetates. The best substrate was 1,2-diacetyl glycerol-3-phosphate with a k_cat/ K_m of 4.9 × 10³ M^-1 s^-1. The presence of a phosphate group in the substrate was essential for enzymatic hydrolysis by the enzyme. It was surprising, however, to find that PHP was unable to hydrolyze any of the lactones tested as potential substrates, unlike most of the other enzymes from cog1735.

Figure 1.

Sequence similarity network representation (http://www.cytoscape.org) of cog1735 from the amidohydrolase superfamily obtained at E-value cutoffs of 10⁻⁷⁰ (a) and 10⁻⁴⁰ (b). Each node in the network represents a single sequence, and each edge (depicted as a line) represents the pairwise connection between two sequences at the given BLAST E-value. The triangular nodes represent proteins that have been functionally and/or structurally characterized. (c) Organization of the php gene cluster in E. coli. The adjacent genes encode proteins that are predicted to be a PLP-dependent racemase (yhfX), a putative sugar isomerase (yhfW), a putative transporter (yhfT) and a putative PLP-dependent lyase (yhfS).

Figure 2.

Cartoon representation of the E. coli PHP (PDB id: 4LEF) with zinc (colored in purple) bound in the active site. The protein is colored based on the secondary structure: α-helices (green), β-strands (maroon) and loops (white). The N-terminus and the C-terminus are labeled as N and C, respectively.

Figure 3.

(a) Coordination geometry of the metal center in the active site of PHP. A phosphate group coordinates with both metal ions. (b) Positioning of the phosphate ligand in the active site of PHP. Metal coordinating histidine residues are not shown for clarity.

Figure 4.

(a) PHP with compound 24 docked in the active site as a high energy intermediate. (b) PHP with 25 (R-enantiomer) docked in the active site as a high energy intermediate. (c) Proposed high-energy intermediates of the hydrolysis reaction catalyzed by PHP. Compound 24 from compound 2 and compound 25 from compound 15.

Scheme 1.

Structures of compounds hydrolyzed by PHP.