Hydrolysis of Phosphodiesters through Transformation of the Bacterial Phosphotriesterase (1998)

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 >108 times faster than the uncatalyzed reaction (kcat = 0.06 s−1 andKm = 38 mm). Upon the addition of various alkylamines to the reaction mixture, thekcat/Km 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 (kcat = 0.34 s−1 and Km = 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 wild-type 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 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 >108 times faster than the uncatalyzed reaction (kcat = 0.06 s−1 andKm = 38 mm). Upon the addition of various alkylamines to the reaction mixture, thekcat/Km 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 (kcat = 0.34 s−1 and Km = 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 wild-type 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 (1Dumas D.P. Caldwell S.R. Wild J.R. Raushel F.M. J. Biol. Chem. 1989; 264: 19659-19665Abstract Full Text PDF PubMed Google Scholar, 2Donarski W.J. Dumas D.P. Heitmeyer D.H. Lewis V.E. Raushel F.M. Biochemistry. 1989; 28: 4650-4655Crossref PubMed Scopus (216) Google Scholar). Paraoxon, the best characterized substrate for the zinc-substituted phosphotriesterase, is hydrolyzed with akcat/Km of 4 × 107m−1 s−1 and akcat of 2100 s−1. The active site of this enzyme contains a coupled binuclear metal center, which is absolutely essential for catalytic activity (1Dumas D.P. Caldwell S.R. Wild J.R. Raushel F.M. J. Biol. Chem. 1989; 264: 19659-19665Abstract Full Text PDF PubMed Google Scholar). The native enzyme contains two Zn2+ ions, but these metal ions can be replaced with Co2+, Ni2+, Mn2+, or Cd2+ with retention of full catalytic activity. From chemical, kinetic, and genetic studies, it has been demonstrated that the reaction proceeds via an Sn2-like associative mechanism in which a metal-bound hydroxide ion attacks the electrophilic phosphorus center of the substrate (2Donarski W.J. Dumas D.P. Heitmeyer D.H. Lewis V.E. Raushel F.M. Biochemistry. 1989; 28: 4650-4655Crossref PubMed Scopus (216) Google Scholar, 3Caldwell S.R. Newcomb J.R. Schlecht K.A. Raushel F.M. Biochemistry. 1991; 30: 7438-7444Crossref PubMed Scopus (163) Google Scholar, 4Caldwell S.R. Raushel F.M. Weiss P.M. Cleland W.W. Biochemistry. 1991; 30: 7444-7450Crossref PubMed Scopus (113) Google Scholar, 5Lewis V.E. Donarski W.J. Wild J.R. Raushel F.M. Biochemistry. 1988; 27: 1591-1597Crossref PubMed Scopus (169) Google Scholar, 6Omburo G.A. Mullins L.S. Raushel F.M. Biochemistry. 1993; 32: 9148-9155Crossref PubMed Scopus (58) Google Scholar, 7Chae M.Y. Omburo G.A. Lindahl P.A. Raushel F.M. J. Am. Chem. Soc. 1993; 115: 12173-12174Crossref Scopus (40) Google Scholar, 8Kuo J.M. Raushel F.M. Biochemistry. 1994; 33: 4265-4272Crossref PubMed Scopus (54) Google Scholar, 9Kuo J.M. Chae M.Y. Raushel F.M. Biochemistry. 1997; 36: 1982-1988Crossref PubMed Scopus (57) Google Scholar, 10Benning M.M. Kuo J.M. Raushel F.M. Holden H.M. Biochemistry. 1994; 33: 15001-15007Crossref PubMed Scopus (197) Google Scholar, 11Benning M.M. Kuo J.M. Raushel F.M. Holden H.M. Biochemistry. 1995; 34: 7973-7978Crossref PubMed Scopus (191) Google Scholar, 12Vanhooke J.L. Benning M.M. Raushel F.M. Holden H.M. Biochemistry. 1996; 35: 6020-6025Crossref PubMed Scopus (246) Google Scholar, 13Dumas D.P. Raushel F.M. J. Biol. Chem. 1990; 265: 21498-21503Abstract Full Text PDF PubMed Google Scholar, 14Banzon J.A. Kuo J.M. Miles B.W. Fischer D.R. Stang P.J. Raushel F.M. Biochemistry. 1995; 34: 743-749Crossref PubMed Scopus (27) Google Scholar, 15Banzon J.A. Kuo J.M. Fischer D.R. Stang P.J. Raushel F.M. Biochemistry. 1995; 34: 750-754Crossref PubMed Scopus (16) Google Scholar, 16Hong S.-B. Raushel F.M. Biochemistry. 1996; 35: 10904-10912Crossref PubMed Scopus (134) Google Scholar). 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 (16Hong S.-B. Raushel F.M. Biochemistry. 1996; 35: 10904-10912Crossref PubMed Scopus (134) Google Scholar). Not surprisingly, the substrate-binding site pocket consists predominantly of hydrophobic residues (12Vanhooke J.L. Benning M.M. Raushel F.M. Holden H.M. Biochemistry. 1996; 35: 6020-6025Crossref PubMed Scopus (246) Google Scholar) that can readily accommodate a variety of nonpolar organophosphate triesters, which explains, in part, the relatively broad substrate specificity of this enzyme. Phosphodiesters are chemically more resistant to hydrolysis than are phosphotriesters. For example, it has been estimated that ethyl-4-nitrophenyl phosphate (StructureFs1, compound I) spontaneously hydrolyzes with kobs ≤ 10−10s−1 at 25 °C and pH 8 (17Hendry P. Sargeson A.M. J. Am. Chem. Soc. 1989; 111: 2521-2527Crossref Scopus (130) Google Scholar), whereas under the same reaction conditions, the base-catalyzed hydrolysis of paraoxon, diethyl-4-nitrophenyl phosphate (Structure Fs1, compound II), proceeds with a rate constant of ∼10−7 s−1(1Dumas D.P. Caldwell S.R. Wild J.R. Raushel F.M. J. Biol. Chem. 1989; 264: 19659-19665Abstract Full Text PDF PubMed Google Scholar). The exceptional chemical stability of the phosphodiester backbone in molecules such as RNA and DNA facilitates the conservation of genetic information. An array of model compounds that promote the catalytic hydrolysis of phosphodiester bonds has been designed and synthesized (17Hendry P. Sargeson A.M. J. Am. Chem. Soc. 1989; 111: 2521-2527Crossref Scopus (130) Google Scholar, 18Morrow J.R. Trogler W.C. Inorg. Chem. 1988; 27: 3387-3394Crossref Scopus (151) Google Scholar, 19Deal K.A. Hengge A.C. Burstyn J.N. J. Am. Chem. Soc. 1996; 118: 1713-1718Crossref Scopus (110) Google Scholar, 20Deal K.A. Burstyn J.N. Inorg. Chem. 1996; 35: 2792-2798Crossref Scopus (131) Google Scholar, 21Hettich R. Schneider H.-J. J. Am. Chem. Soc. 1997; 119: 5638-5647Crossref Scopus (253) Google Scholar, 22Han M.J. Yoo K.S. Cho T.J. Chang J.Y. Cha Y.J. Nam S.H. J. Chem. Soc. Chem. Commun. 1997; : 163-164Crossref Scopus (10) Google Scholar, 23Bunton C.A. Diaz S. Hellyer J.M. Ihara Y. Ionescu L.G. J. Org. Chem. 1975; 40: 2313-2317Crossref Scopus (21) Google Scholar). Nearly all of the model compounds constructed to date utilize metal ions as essential cofactors to activate the substrate and nucleophile while holding the reactants together in close proximity (17Hendry P. Sargeson A.M. J. Am. Chem. Soc. 1989; 111: 2521-2527Crossref Scopus (130) Google Scholar, 18Morrow J.R. Trogler W.C. Inorg. Chem. 1988; 27: 3387-3394Crossref Scopus (151) Google Scholar, 19Deal K.A. Hengge A.C. Burstyn J.N. J. Am. Chem. Soc. 1996; 118: 1713-1718Crossref Scopus (110) Google Scholar, 20Deal K.A. Burstyn J.N. Inorg. Chem. 1996; 35: 2792-2798Crossref Scopus (131) Google Scholar, 21Hettich R. Schneider H.-J. J. Am. Chem. Soc. 1997; 119: 5638-5647Crossref Scopus (253) Google Scholar). However, the enzymatic hydrolysis of the phosphodiester bond is typically much faster than the small molecule mimics. Many enzymes are known to catalyze the hydrolysis of the phosphodiester bond, with kcat values ranging from 10−2 to 103 s−1. This class of enzymes includes, for example, the hammerhead ribozyme, which catalyzes the site-specific hydrolysis of a phosphodiester bond withkcat ∼ 1 min−1 (24Birikh K.R. Heaton P.A. Eckstein F. Eur. J. Biochem. 1997; 245: 1-16Crossref PubMed Scopus (243) Google Scholar); ribonuclease A (kcat = 1400 s−1 for UpA) (25delCardayré S.B. Raines R.T. Biochemistry. 1994; 33: 6031-6037Crossref PubMed Scopus (110) Google Scholar); ribonuclease T1 (kcat = 350 s−1 for GpC) (26Takagashi K. Moore S. Boyer P.D. The Enzymes. 3rd Ed. Volume XV. Academic Press, New York1982: 458-461Google Scholar); EcoRI endonuclease (kcat = 0.9 s−1 for GpAATTC) (27Kurpiewski M.R. Koziolkiewicz M. Wilk A. Stec W.J. Jen-Jacobson L. Biochemistry. 1996; 35: 8846-8854Crossref PubMed Scopus (30) Google Scholar); and staphylococcal nuclease (kcat = 150 s−1 for calf thymus DNA) (28Alexander M. Heppel L.A. Hurwitz J. J. Biol. Chem. 1961; 236: 3014-3019Abstract Full Text PDF PubMed Google Scholar). The existence of phosphotriesters in nature is decidedly quite rare, and thus, it is still unclear as to the origin of the metabolic pressure for the selection and enhancement of the enormous catalytic activity exhibited by the bacterial phosphotriesterase from P. diminuta. Since the catalytic machinery required for the hydrolysis of phosphodiesters is apparently similar to the binuclear metal center found within the active site of phosphotriesterase, we anticipated that phosphotriesterase would possess an inherent ability to hydrolyze phosphodiester substrates. However, the active site of phosphotriesterase is filled predominantly with hydrophobic residues, and thus, this site may not be suitable for the accommodation of the negatively charged phosphodiesters. In this paper, we provide direct experimental evidence to support the conclusion that the bacterial phosphotriesterase has a significant amount of phosphodiesterase activity. Moreover, the rate enhancement can be intensified by the incorporation of a positive charge within the active site, either through site-directed mutagenesis or the addition of alkylamines to the aqueous medium. The bacterial phosphotriesterase and the metal-substituted derivatives were isolated as described previously (29Omburo G.A. Kuo J.M. Mullins L.S. Raushel F.M. J. Biol. Chem. 1992; 267: 13278-13283Abstract Full Text PDF PubMed Google Scholar). The site-directed mutagenesis of the phosphotriesterase variants (M317A, M317H, M317K, and M317R) was carried out as described previously (8Kuo J.M. Raushel F.M. Biochemistry. 1994; 33: 4265-4272Crossref PubMed Scopus (54) Google Scholar, 9Kuo J.M. Chae M.Y. Raushel F.M. Biochemistry. 1997; 36: 1982-1988Crossref PubMed Scopus (57) Google Scholar, 30Watkins L.M. Kuo J.M. Chen-Goodspeed M. Raushel F.M. Proteins Struct. Funct. Genet. 1997; 29: 553-561Crossref PubMed Scopus (14) Google Scholar). Cell growth and purification of the mutant enzymes were performed according to published procedures (29Omburo G.A. Kuo J.M. Mullins L.S. Raushel F.M. J. Biol. Chem. 1992; 267: 13278-13283Abstract Full Text PDF PubMed Google Scholar). All of the chemicals used in these experiments were purchased from Sigma, Aldrich, Fisher, or U. S. Biochemical Corp. Biochemical supplies were purchased from Promega, Amersham Pharmacia Biotech, Bio 101, Inc., Perkin-Elmer, or Hoffmann-La Roche. The synthesis of oligonucleotides and DNA sequencing reactions were carried out in the Gene Technology Laboratory of the Biology Department at Texas A&M University. Compound I was synthesized according to the procedure of Hendry and Sargeson (17Hendry P. Sargeson A.M. J. Am. Chem. Soc. 1989; 111: 2521-2527Crossref Scopus (130) Google Scholar). Compounds III–VI (StructureFs2) were synthesized by a similar procedure with the following modifications. Ethyl dichlorophosphate (12.3 mmol, 1.46 ml) was mixed with triethylamine (12.3 mmol, 1.72 ml) in dry ethyl ether (10 ml). One equivalent of the substituted phenol, dissolved in dry ethyl ether (20 ml), was added dropwise to this solution over a period of 30 min at 0 °C. The mixture was stirred for 1 h; triethylamine (1.72 ml) was added to the mixture; and the stirring was continued for an additional 15 min. Finally, water (5 ml) was added, and the mixture was stirred for 30 min. The product was extracted three times with 30-ml portions of a solution of 0.2m aqueous triethanolamine and the water was in The was in and the was by The solution was over and the was The product was dissolved in and with 30 of to the The was over and the was in to the The of were by were made a The reactions were by the of the substituted from the hydrolysis of the phosphodiester the addition of the phosphotriesterase at 25 °C. The at pH and values of the were from the S.R. Newcomb J.R. Schlecht K.A. Raushel F.M. Biochemistry. 1991; 30: 7438-7444Crossref PubMed Scopus (163) Google Scholar). at pH values were 1 is the of the substituted of the various amines were made at and pH values were or The values of kcat andKm were by the to is the is the is the and A is the substrate The were by the to to the 4 The were to and the values were from the of these The from the the phosphodiester was at of were to A is the of is the of Km is the constant of the phosphodiester in the absence of added is the in the absence of added is the constant for the of an and and are the of Km in the presence and absence of added Compound I was as a substrate for the bacterial The rate of hydrolysis of compound I catalyzed by the phosphotriesterase was (kcat = 0.06 = s−1 at pH with that of paraoxon (kcat = = × 107m−1 s−1 at pH but was >108 times faster than the uncatalyzed reaction under similar reaction The phosphotriesterase used for this was by The phosphodiesterase and phosphotriesterase for compounds I were for the purification in addition to the at A of these the is in 1 and to the of the two the Four mutant enzymes were also in a preliminary attempt to the rate of phosphodiesterase activity exhibited by the native enzyme. the mutant M317K catalyzed the hydrolysis of the phosphodiester substrate (compound I) with the (kcat = 0.34 s−1 = s−1 at pH The for the (M317A, and are in for the mutant enzymes with phosphodiester and × × × × × were pH and 25 °C for the The were to and the for of the were of the in a were pH and 25 °C for the The were to and the for of the were of the The hydrolysis rate of compound I was up to various alkylamines were added to the reaction The for these rate are in In a rate was for the wild-type enzyme amines with were amines showed a enhancement than amines. The rate enhancement was with (kcat = s−1 = s−1 at pH For the M317A mutant amines with was the best for this mutant enzyme (kcat = s−1 = s−1 at pH enhancement of the phosphodiesterase activity of the wild-type phosphotriesterase was in the presence of or at of of added amines on phosphodiester (compound I) hydrolysis by of used to the phosphodiesterase activity of were at 25 °C and pH The were to The for of the were of the of used to the phosphodiesterase activity of in a of added amines on the phosphodiester hydrolysis by the M317A mutant of used to the phosphodiesterase activity of M317A were at 25 °C and pH The were to The for of the were of the of used to the phosphodiesterase activity of M317A in a These were at 25 °C and pH The were to The for of the were of the These were at 25 °C and pH The were to The for of the were of the The of pH were made added and also with either or in the reaction In the reactions were and values were was added, and thus, values be with In the pH added it can be that of a the catalytic reaction. The of this not be from the experimental over the pH range of was added, of a the reaction rate. From a of the to the of this was to be are involved in the enzymatic reaction in the presence of of the to values of and for these two The for phosphodiester hydrolysis by the and mutant enzymes were also The of pH for enzymes a in activity pH not From a of these to values of and were for the and The of of the binuclear metal center of the wild-type phosphotriesterase with Co2+, and Cd2+ at pH and are in The for paraoxon (compound hydrolysis are for The enzyme the kcat for phosphodiester (compound I) hydrolysis as as for paraoxon The of values Cd2+ at pH is similar to that of paraoxon of phosphodiester (compound I) and paraoxon by the metal-substituted pH not be the reaction were to were at 25 °C. The were to The are of the These not be the reaction were to in a These were at 25 °C. The were to The are of the The added and phosphodiester were together in to the mechanism for the enhancement reaction. The reaction rate was increased with of and these were to The values of kcat andKm are 0.06 s−1 and 38 The and values are and These values demonstrate that the addition of the and were for compounds I in the presence and absence of was and amines or with the of and In the absence of added values be the Km values were to be The a the values of the and or The values are and for in the absence and presence of added The in the presence of added is The full negative charge within the phosphodiester substrate is thought to be responsible for the rate of catalytic hydrolysis of these compounds by the The active site of the phosphotriesterase is hydrophobic (12Vanhooke J.L. Benning M.M. Raushel F.M. Holden H.M. Biochemistry. 1996; 35: 6020-6025Crossref PubMed Scopus (246) Google Scholar), and thus, it would not be to accommodate the negative charge on the substrate the nucleophile in the active site, hydroxide may not be to attack the substrate a the phosphodiesters and Km the hydrolytic reaction were added to the reaction in an attempt to the negative charge of the were are in the pH range used for this and the and be by the and the of the For the wild-type faster reaction were amines with were added, whereas the M317A mutant enzyme amines with is within the pocket of the enzyme active site (12Vanhooke J.L. Benning M.M. Raushel F.M. Holden H.M. Biochemistry. 1996; 35: 6020-6025Crossref PubMed Scopus (246) Google Scholar). It was thought that by the to a amines be more with the reaction faster than amines with and amines were more than amines in the reaction. The for these are not The three mutant M317H, M317K, and were designed to a within the enzyme active site amines from the medium. The rate were not and the showed a similar to that of the wild-type enzyme added It that these mutant enzymes catalyze the phosphodiesterase reaction with from the substituted residues, with the of M317K, which showed a enhancement The small rate enhancement by these in part, be to the and a substrate in the enzyme active site The the of the of and the phosphorus center of the substrate is (12Vanhooke J.L. Benning M.M. Raushel F.M. Holden H.M. Biochemistry. 1996; 35: 6020-6025Crossref PubMed Scopus (246) Google Scholar). this is replaced with residues with and the the positive charge of the and the negative charge of a phosphodiester substrate was estimated to be to model the of the substrate and mutated Many been made to mutant enzymes with substrate For example, the of that ion with the of the substrate was mutated to in an attempt to the substrate specificity of this enzyme from to the and of of the for and were J. Chem. Soc. Scopus Google Scholar). In a the same thekcat/Km for the of was increased of residues 1995; PubMed Scopus Google Scholar). The pH for hydrolysis in the absence of added that a to be for catalytic activity. This be either the of the phosphodiester substrate or the metal-bound hydroxide The of the substrate would the reaction by the negative charge of the substrate and it more a the of the metal-bound hydroxide would the of the but charge would be and the reaction rate may be The in the presence of a that are two involved in the catalytic reaction. One of these may be the metal-bound water = whereas the may be = The reaction rate at the hydroxide is and also at high the is The pH in the presence of a The rate as the pH it is at pH The of the is and from this and the of the it is thought that this is the metal-bound water molecule (16Hong S.-B. Raushel F.M. Biochemistry. 1996; 35: 10904-10912Crossref PubMed Scopus (134) Google Scholar). the with the rate not at high pH cation be pH The pH of the and mutant enzymes for phosphodiester hydrolysis were also The of these were similar to the one with the wild-type enzyme reaction added The a of activity at high whereas the of the wild-type enzyme in the presence of a of activity at It that the of the mutant enzyme is not for charge and the reaction proceeds significant from the The relatively small enhancement of thekcat/Km this were made at in an to the mechanism of the enzymatic reaction. The and the are shown in to which the were was from the mechanism shown in this the a with the and the substrate to an This is hydrolyzed to the of the = the reaction mechanism a reaction with 0.06 s−1 and Km = 38 The has a faster = and a Km = than the An mechanism in which a is to to the active site not the experimental The for the of on the the in the absence and presence of values of and The negative values that the chemical is the and the of the reaction has In the presence of the of of the has a of and was more negative than the of the of the same reaction The in this demonstrate that the catalytic machinery that has for the hydrolysis of phosphotriesters can be to hydrolyze phosphodiester substrates. The the phosphotriesterase has in of phosphodiester is in accommodation of the negative charge and the of from the These in part, be through chemical via the addition of alkylamines from the medium. to the same through site-directed mutagenesis were The most likely for this is the of the positive charge from the mutant is to and mutant enzymes that can more catalyze the hydrolysis of phosphodiester A model for phosphodiester hydrolysis as catalyzed by the bacterial phosphotriesterase is in The phosphoryl oxygen bond is by the metal ions, and the negative charge is by the added The mechanism is similar to that of the hydrolysis (16Hong S.-B. Raushel F.M. Biochemistry. 1996; 35: 10904-10912Crossref PubMed Scopus (134) Google Scholar), the metal ions the phosphoryl oxygen bond and the metal-bound hydroxide attacks the phosphorus