Hydrolysis of Phosphodiesters through Transformation of the Bacterial Phosphotriesterase

Abstract

The phosphotriesterase from Pseudomonas diminuta catalyzes the hydrolysis of a wide array of phosphotriesters and related phosphonates, including organophosphate pesticides and military nerve agents. It has now been shown that this enzyme can also catalyze the hydrolysis of phosphodiesters, albeit at a greatly reduced rate. However, the enzymatic hydrolysis of ethyl-4-nitrophenyl phosphate (compound I) by the wild-type enzyme was >10 8 times faster than the uncatalyzed reaction (k cat ‫؍‬ 0.06 s ؊1 and K m ‫؍‬ 38 mM). Upon the addition of various alkylamines to the reaction mixture, the k cat /K m for the phosphodiester (compound I) increased up to 200-fold. Four mutant enzymes of the phosphotriesterase were constructed in a preliminary attempt to improve phosphodiester hydrolysis activity of the native enzyme. Met-317, which is thought to reside in close proximity to the pro-S-ethoxy arm of the paraoxon substrate, was mutated to arginine, alanine, histidine, and lysine. These mutant enzymes showed slight improvements in the catalytic hydrolysis of organophosphate diesters. The M317K mutant enzyme displayed the most improvement in catalytic activity (k cat ‫؍‬ 0.34 s ؊1 and K m ‫؍‬ 30 mM). The M317A mutant enzyme catalyzed the hydrolysis of the phosphodiester (compound I) in the presence of alkylamines up to 200 times faster than the wildtype enzyme in the absence of added amines. The neutralization of the negative charge on the oxygen atom of the phosphodiester by the ammonium cation within the active site is thought to be responsible for the rate enhancement by these amines in the hydrolytic reaction. These results demonstrate that an active site optimized for the hydrolysis of organophosphate triesters can be made to catalyze the hydrolysis of organophosphate diesters.The bacterial phosphotriesterase from Pseudomonas diminuta catalyzes the hydrolysis of a wide range of organophosphate nerve agents with high efficiency (1, 2). Paraoxon, the best characterized substrate for the zinc-substituted phosphotriesterase, is hydrolyzed with a k cat /K m of 4 ϫ 10and a k cat of 2100 s Ϫ1 . The active site of this enzyme contains a coupled binuclear metal center, which is absolutely essential for catalytic activity (1). The native enzyme contains two Zn 2ϩ ions, but these metal ions can be replaced with Co 2ϩ , Ni 2ϩ, Mn 2ϩ , or Cd 2ϩ with retention of full catalytic activity. From chemical, kinetic, and genetic studies, it has been demonstrated that the reaction proceeds via an S n 2-like associative mechanism in which a metal-bound hydroxide ion attacks the electrophilic phosphorus center of the substrate (2-16). The role of one of the two metal ions within the active site is thought to involve the activation of the hydrolytic water molecule, whereas the companion metal ion is most likely involved in the polarization of the phosphoryl oxygen bond of the substrate to increase the electrophilicity of the substrate for nucleophilic attack (16). Not surprisingly, the substrate-binding site pocket consists predominantly of ...

Functional Interrelationships in the Alkaline Phosphatase Superfamily: Phosphodiesterase Activity of Escherichia coli Alkaline Phosphatase

“…Recent findings that other two metal ion catalysts have promiscuous phosphodiesterase activity provide additional evidence for the inherent catalytic potential of binuclear clusters for catalyzing phosphate diester hydrolysis. The bacterial phosphotriesterase (68), an aminopeptidase from Streptomyces (69), and now alkaline phosphatase have each been shown to provide substantial rate enhancements for cleavage of a phosphate diester bond [(k cat /K m )/k w ) 10 10 - 10 15 ]. As each of these enzymes catalyzes different chemical transformations in their physiological roles and has independent evolutionary histories, this suggests that two metal ions positioned ∼4 Å apart are especially conducive to catalysis of phosphate diester hydrolysis.…”

ElaC Encodes a Novel Binuclear Zinc Phosphodiesterase

“…A potential physiological phosphodiester substrate for phosphonate monoester hydrolase, however, has not yet been found. An interesting fact is that other binuclear zinc enzymes with an established physiological function, such as aminopeptidase (33), phosphotriesterase (34), and alkaline phosphatase (phosphoric acid monoester hydrolase) (35), also display activity to artificial phosphodiesters (Table IV). Although the activity is orders of magnitude lower as compared with that of ZiPD, this shows that a binuclear zinc active site is preferred for phosphodiester cleavage.…”

Cloning and expression of the phosphotriesterase gene hocA from Pseudomonas monteilii C11 b bThe GenBank accession number for the hocA gene is AF469117.

“…While this suggests a role for HocA in phosphate metabolism, it is highly unlikely that the native role is to hydrolyse phosphotriesters, because OPs have only been in existence for the last 50 years. HocA may have evolved from a pre-existing phosphatase or phosphodiesterase as it has been shown that a phosphotriesterase (OPH) could be altered by only one amino acid to possess phosphodiesterase activity (Shim et al, 1998), demonstrating how a minor change can allow a protein to acquire a new activity. Likewise, HocA may have been a pre-existing phosphodiesterase that has acquired phosphotriesterase activity.…”