Multiple Reaction Products from the Hydrolysis of Chiral and Prochiral Organophosphate Substrates by the Phosphotriesterase from <i>Sphingobium</i> sp. TCM1

The phosphotriesterase from <i>Sphingobium</i> sp. TCM1 (<i>Sb</i>-PTE) is notable for its ability to hydrolyze organophosphates that are not substrates for other enzymes. In an attempt to determine the catalytic properties of <i>Sb</i>-PTE for hydrolysis of chiral phosphotriesters, we discovered that multiple phosphodiester products are formed from a single substrate. For example, <i>Sb</i>-PTE catalyzes the hydrolysis of the <i>R</i>_P-enantiomer of methyl cyclohexyl <i>p</i>-nitrophenyl phosphate with exclusive formation of methyl cyclohexyl phosphate. However, the enzyme catalyzes hydrolysis of the <i>S</i>_P-enantiomer of this substrate to an equal mixture of methyl cyclohexyl phosphate and cyclohexyl <i>p</i>-nitrophenyl phosphate products. The ability of this enzyme to catalyze the hydrolysis of a methyl ester at the same rate as the hydrolysis of a <i>p</i>-nitrophenyl ester contained within the same substrate is remarkable. The overall scope of the stereoselective properties of this enzyme is addressed with a library of chiral and prochiral substrates

Chemical Mechanism of the Phosphotriesterase from <i>Sphingobium</i> sp. Strain TCM1, an Enzyme Capable of Hydrolyzing Organophosphate Flame Retardants

The mechanism of action of the manganese-dependent phosphotriesterase from <i>Sphingobium</i> sp. strain TCM1 that is capable of hydrolyzing organophosphate flame retardants was determined. The enzyme was shown to hydrolyze the <i>R</i>_P-enantiomer of <i>O</i>-methyl <i>O</i>-cyclohexyl <i>p</i>-nitrophenyl thiophosphate with net inversion of configuration and without the formation of a covalent reaction intermediate. These results demonstrate that the enzyme catalyzes the hydrolysis of substrates by activation of a nucleophilic water molecule for direct attack at the phosphorus center

Structure-Based Function Discovery of an Enzyme for the Hydrolysis of Phosphorylated Sugar Lactones

Two enzymes of unknown function from the cog1735 subset of the amidohydrolase superfamily (AHS), LMOf2365_2620 (Lmo2620) from <i>Listeria monocytogenes str.</i> 4b F2365 and Bh0225 from <i>Bacillus halodurans</i> C-125, were cloned, expressed, and purified to homogeneity. The catalytic functions of these two enzymes were interrogated by an integrated strategy encompassing bioinformatics, computational docking to three-dimensional crystal structures, and library screening. The three-dimensional structure of Lmo2620 was determined at a resolution of 1.6 Å with two phosphates and a binuclear zinc center in the active site. The proximal phosphate bridges the binuclear metal center and is 7.1 Å from the distal phosphate. The distal phosphate hydrogen bonds with Lys-242, Lys-244, Arg-275, and Tyr-278. Enzymes within cog1735 of the AHS have previously been shown to catalyze the hydrolysis of substituted lactones. Computational docking of the high-energy intermediate form of the KEGG database to the three-dimensional structure of Lmo2620 highly enriched anionic lactones versus other candidate substrates. The active site structure and the computational docking results suggested that probable substrates would likely include phosphorylated sugar lactones. A small library of diacid sugar lactones and phosphorylated sugar lactones was synthesized and tested for substrate activity with Lmo2620 and Bh0225. Two substrates were identified for these enzymes, d-lyxono-1,4-lactone-5-phosphate and l-ribono-1,4-lactone-5-phosphate. The <i>k</i>_cat/<i>K</i>_m values for the cobalt-substituted enzymes with these substrates are ∼10⁵ M^–1 s^–1

Functional Annotation and Structural Characterization of a Novel Lactonase Hydrolyzing d‑Xylono-1,4-lactone-5-phosphate and l‑Arabino-1,4-lactone-5-phosphate

A novel lactonase from <i>Mycoplasma synoviae</i> 53 (MS53_0025) and <i>Mycoplasma agalactiae</i> PG2 (MAG_6390) was characterized by protein structure determination, molecular docking, gene context analysis, and library screening. The crystal structure of MS53_0025 was determined to a resolution of 2.06 Å. This protein adopts a typical amidohydrolase (β/α)₈-fold and contains a binuclear zinc center located at the C-terminal end of the β-barrel. A phosphate molecule was bound in the active site and hydrogen bonds to Lys217, Lys244, Tyr245, Arg275, and Tyr278. Both docking and gene context analysis were used to narrow the theoretical substrate profile of the enzyme, thus directing empirical screening to identify that MS53_0025 and MAG_6390 catalyze the hydrolysis of d-xylono-1,4-lactone-5-phosphate (<b>2</b>) with <i>k</i>_cat/<i>K</i>_m values of 4.7 × 10⁴ and 5.7 × 10⁴ M^–1 s^–1 and l-arabino-1,4-lactone-5-phosphate (<b>7</b>) with <i>k</i>_cat/<i>K</i>_m values of 1.3 × 10⁴ and 2.2 × 10⁴ M^–1 s^–1, respectively. The identification of the substrate profile of these two phospho-furanose lactonases emerged only when all methods were integrated and therefore provides a blueprint for future substrate identification of highly related amidohydrolase superfamily members