PK Mohapatra.pmd

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ISSN: 0974 - 0376

Save Nature to Survive

: Special issue, Vol. III: 23 - 26 AN INTERNATIONAL QUARTERLY JOURNAL OF ENVIRONMENTAL SCIENCES

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ORIGIN, EVOLUTION AND DIVERSITY OF PHOSPHOTRIESTERASES - AN ORGANOPHOSPHATE DEGRADING ENZYME P. K. Mohapatra and Suchismita Pattanaik

KEYWORDS Organophosphates Phosphotriesterase Enzyme structure Homology Phosphotriesterase like lactonases Phosphotriesterase homology proteins.

Prof. P. C. Mishra Felicitation Volume Paper presented in National Seminar on Ecology Environment & Development 25 - 27 January, 2013 organised by Deptt. of Environmental Sciences, Sambalpur University, Sambalpur 1

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QUARTERLY

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P. K. MOHAPATRA* AND SUCHISMITA PATTANAIK Biotechnology Wing, Department of Botany, Ravenshaw University Cuttack – 753 003, INDIA E-mail:

ABSTRACT Organophosphorus (OP) insecticides are nerve poisons causing neural disco-ordination in the transmission of nerve impulse in target insects. However, the insecticides also impose a variety of nontarget toxicity, thus drawing the attention for research on accelerated degradation of the residues. The OPs are primarily degraded by three important pathways, viz; 1) reduced glutathione dependant decarboxylation or dearylation, 2) cytochrome P 450 mediated oxidation and 3) enzyme catalyzed deesterification. The later is the most efficient mode of degradation of OPs in the field catalyzed by bacterial phosphotriesterases (PTE; EC 3.1.8.1.), an enzyme with unknown physiological role. PTE from Pseudomonas diminuta and Flavobacterium sp. is roughly a globular structure with overall dimensions of approximately 51 Å x 55 Å x 51 Å. It is a dimer that packed with the unit cell with its symmetry axis coincident to the crystallographic dyad running parallel to z axis. It possesses a binuclear metal centre (Zn2+) with a stable structure obtained by interaction among His residues. PTE is believed to have originated in bacteria over the past six decades most probably from a related protein with hydrolytic activity. Three lactonases dubbed PTE like lactonases (PLL) possess both lactone and OP hydrolyzing activities suggesing that PLL gradually evolved to recognise and metabolise OPs. Sequence homology has identified phosphotriesterase homology proteins (PHP), in bacteria which show identical binuclear metal centre and conserved amino acid residues at the active site same as that of PTE. This suggest that PHP might have been the ancestor of PTE, or diverged from a common progenitor, most probably of a PLL. The diversity of PTE and their future application are discussed.

*Corresponding author

INTRODUCTION Organophosphorus (OP) insecticides are anticholinesterase agents imposing their target toxicity through irreversible phosphorylation of a serine residue at the active site of the enzyme acetylcholinesterase (EC 3.1.1.7). The later is the most common enzyme inhibited by OPs but carboxylesterases and butyrilesterases have also been reported to be inhibited by the OP chemicals (Ecobichon, 2001; Corbett, 1974; Dumas et al., 1989; Raushel, 2002). Many other esterases have also been reported to be more or less inhibited by the toxicity of the OP insecticides (Benning et al., 1994). The target action of OP insecticides causes neural discoordination of the pests and abnormal transmission of nerve impulse. However, the insecticides also impose a variety of non-target toxicity to the organisms in the field, mostly by the residual concentrations built up by repeated use of the chemicals. Current research focuses on the accelerated degradation of the OP insecticide residues in the field to shorten the residence of the parent molecules and minimize their non-target toxicity (Li et al., 1995; Cheng et al., 1998; Mohapatra and Schiewer, 2000; Yugui et al., 2008; Singh et al., 2011, Hayasaka et al., 2012). Biodegradation of insecticides plays an important role in regulating the residual concentrations of insecticides in the field and in determining the efficiency of their application in pest control operations. Biodegradation of OP chemicals by different organisms has been reported much before the exact mechanism of the process was developed. Subsequently several types of enzymes were found to be involved in the degradation of OPs to less toxic and non-toxic metabolites. Research evidences suggest that OP chemicals are mostly degraded by three important pathways; viz - 1) Reduced glutathione (GSH) dependent decarboxylation or dearylation (Vontas et al., 2000; Yugui et al., 2008), 2) Cytochrome P450 monooxygenase mediated microsomal oxidation (Nelson, 1999; Singh et al., 2011) and 3) Enzyme catalyzed deesterification (Munnecke, 1976; Dumas et al., 1990a, b; Lai et al., 1994; Benning et al., 1994, 2000; Li et al., 1995; Nelson, 1999; Vontas et al., 2000;Yugui et al., 2008; Baek et al., 2010). Applenton and Nakatsugawa (1972) observed that deesterification was an effective pathway of degradation of OP insecticides and such hydrolytic breakdown was found to be catalyzed by esterases, which do not have any natural substrate. The enzymes are now popularly called as phosphotriesterases (PTEs). Since its discovery a lot of information has been gathered on the origin, structure, catalytic activity, evolution, specificity and application of the enzymes but there is hardly any literature to give in-depth information about this novel enzyme. This review discusses the origin, evolution and diversity of the PTEs and their role in human welfare. Origin and Evolution Several enzymes are involved in deesterification of OPs. They are known from a variety of tissues of mammals, fishes, birds, mulluscs, bacteria and fungi (Vilanova and Sogorb, 1999). These enzymes are called in different names like phosphotriesterase (PTE), organophosphorus hydrolases (OPases/ OPH), 2

PHARMACOLOGICAL EFFICACY OF MEDICINAL PLANTS

organophosphate degrading enzymes (OPD), patathion hydrolases / paraoxonases (PON), OP acid anhydrases (OPAA) and diisopropyl phosphorofluoridases (DFPases) (Vilanova and Sogorb 1999; Scharff et al., 2001; Porzio et al., 2007). The most important among them is the esterase of an unknown physiological role classified by International Union of Biochemistry (IUB) as PTE (EC 3.1.8.1; Dumas et al., 1989). The later is believed to have originated through induced mutation caused by the extensive use of the anthropogenic OP chemicals. The synthesis of paraoxon was first reported in 1950 (Schrader, 1950) though the organophosphate nerve agents were believed to be synthesized during the World War II and the use of these chemicals as agricultural insecticides were used in 1940s (Mazur, 1946). This suggests that the PTE as an enzyme, preferentially using triesters as substrates, originated in bacteria over the past six decades, most probably from a related protein with hydrolytic activity (Vilanova and Sogorb, 1999; Sogorb et al., 1996, 2004). This is supported by the fact that PTE catalyzes the hydrolysis of the insecticide paraoxon at a rate approaching the diffusion limit and thus appears to be optimally evolved for utilizing this synthetic substrate (Dumas et al., 1989; 1990a, b). Since there is no natural triester, which can serve as a substrate for PTE, the origin of this enzyme is certainly caused by induced mutation through prolonged exposure to OP chemical. The most common hypothesis on the origin of bacterial PTE is through evolution from a preexisting hydrolase as a response to the changing environmental conditions, most probably from an enzyme with esterase activity (Dumas et al., 1990b; Buchbinder et al., 1998; Raushel and Holden, 2000; Raushel, 2002; Roodveldt and Tawfik, 2005). PTE was found to have promiscuous esterase and lactonase activity. At least three more lactonases, dubbed PTE like lactonases (PLL) possess both lactonase and organophosphate activities (Afriat et al., 2006). Compared to PTEs, PLLs hydrolyze a broad range of lactones but their OP hydrolyzing activities are much lower. While PLLs are more or less specific in using lactones as substrates, PTE is a metalloenzyme with ability to hydrolyze a broad range of organophosphorus compounds, including the chemical warfare agents and agricultural pesticides (Omburo et al., 1992; Benning, et al., 1994, 1995, Lai et al., 1995; Masson et al., 1998). This suggests that PLL gradually evolved to shed their affinities to lactones and more efficiently recognized and metabolized the organophosphorus triesters. The phosphotriesterase from Flavobacterium was first identified from samples taken from a Philippine rice patty treated with the organophosphate diazinon as a means of insect control (Sethunathan and Yoshida, 1973). At about the same time, a strain of the soil bacterium Pseudomonas diminuta was isolated through its ability to hydrolyze parathion (Munnecke, 1976). The genes encoding the active enzymes (opd, for organophos phate - degrading) were localized to extrachromosomal plasmids in both the cases. The gene sequences for the enzyme from the two sources were identical. Database searches identified other proteins that appear to be close relatives of PTE. However, the ability of these proteins to function as authentic phosphotriesterases has not, as yet, been determined. The closest sequence homologue of PTE identified to date is Phosphotriesterase Homology Protein (PHP), an enzyme with unknown function.

PHP is a member of amidohydrolase superfamily and exhibits 28% sequence identity, and 66% sequence similarity to PTE (Roodveldt and Tawfik, 2005). Both the enzymes possess an essentially identical binuclear metal (Zn2+) centre and residues involved in their coordination are well conserved. In addition thir overall structure show excellent superposition (Lai et al., 1994; Roodveldt and Tawfik, 2005). However, PHP differ from PTE in catalytic activity and does not catalyze phosphotriester hydrolysis. Besides E. coli PHP had no enzymatic activity in the presence of general esterase and PTE substrates (Yildirim et al., 2005). But subsequent studies reported a weak esterase activity and PTE activity in an E. coli PHP mutant (Yildirim et al., 2009). These finding suggested that PHP might have been the ancestor of PTE, or that these enzymes diversed from a common progenitor, most probably of a lactonase type enzyme and evolved, by induction, into an OP degrading enzyme (Scalan and Reid, 1995). Diversity OP hydrolyzing enzymes are known from several different microbial species. Different class of enzymes able to degrade more specifically nerve gases are identified from Pseudomonas diminuta and Flavobacterium sp. (Dumas et al., 1989), Alteromonas (Cheng et al., 1993; 1998, Vyas et al., 2010), Aspergillus niger (Liu et al., 2001) other Psuedomonads, E. coli, Sulfolobus spp. (Porzio et al., 2007) and Pyrococcus sp. (Theriot et al., 2010a,b). PHP identified from E. coli represents archetypical type of enzymes from which PTEs have certainly evolved (Hou et al., 1996) and the protein show paraoxonase activity on mutation (Roodveldt and Tawfik, 2005). A protein from higher thermophilic archeon Sulfolobus solfataricus and Sulfolobus acidocaldarius also showed OP hydrolyzing activity and was considered as a new type of PTE (Porzio et al., 2007). The enzyme has hydrolytic activity against PNP-butanoate, bis-PNP-phosphate and several OP insecticides. OP resistance associated mutations have been reported in insects resulting in the reduction in sensitivity of the insects through enzyme mediated degradation of insecticides (Tsagkarakou et al., 2009). Similarly enzymatic hydrolysis of specific OP chemicals have been reported in fungi, insects, mammals and higher plants (Liu et al., 2001; Scharff et al., 2001; Khillar et al., 2010) indicating a highly diverse OP hydrolyzing enzymes distributed in plants, animals and microbes. PTEs are located in several tissues of mammals. Higher levels of PTEs activities are typically found in serum and liver (Vilanova and Sogorb, 1999). Hydrolysis by PTEs is considered a detoxifying reaction. Fig. 1 displays well known reactions catalysed by PTEs. The PTE best characterised is an activity called ‘paraoxonase’. This hydrolyses the insecticide paraoxon (O, O-diethyl-pnitrophenyl - phosphate) and it has been studied mainly in the plasma of mammals and in the bacterium Pseudomonas diminuta (Dumas et al., 1989, 1990a, b). Usually, PTEs are called along with the name of their substrate. For example the enzymes that hydrolyse paraoxon, diisopropylphos phorofluoridate (DFP) and O-hexyl O -2, 5 - dichlorophenyl phosphoradate (HDCP) are called paraoxonase (and also PTE) (EC 3.1.8.1), DFPase (EC 3.1.8.2) and HDCPase (EC 3.1.8.3), respectively (Fig. 1). The latter were further subdivided into Mazur-type DFPases (40–96 kDa) and squid type DFPases (35–40 kDa (Serdar et al., 1989). Squid-type DFPases hydrolyze DFP 2–20 times faster than soman. Human serum paraoxonase 3

P. K. MOHAPATRA AND SUCHISMITA PATTANAIK

This consequently proves that PTE has also the active site in the C- terminal portion of barrel. Benning et al. (1994) have demonstrated that the interaction among the His residues in PTE is instrumental in the structural stability and catalytic activity of the enzyme. There is a stacking interaction between the imidazole rings of His 201 and His 254. In addition N ä1 of His 254 lies at 2.5 Å from Oä1 of Asp 253 (Table 1). This acidic residue, in turn, participates in the electrostatic interaction with side chain of His 55 thus serving as a bridge between His 254 and His 55. There is a very close contact between His 201 and Lys 169. There are two aromatic residues located within the vicinity of the His cluster. The carbonyl oxygen of His 230 lies at 3.3Aº from N ä1 of Trp 302 while N å2 of its imidazole side chain forms a hydrogen bond with the hydroxyl group of Tyr 239 (Benning et al., 1994, 1995, 2000, 2001; Raushel, 2002). Initial evidences on the structure of PTE were presenting it as a monomer. But from the aromatic contacts and numerous electrostatic interactions occurring along the interface between the symmetry related molecules of the enzyme Benning et al. (1994, 1995) proved that PTE is a dimer that packed with the unit cell with its symmetry axis coincident to the crystallographic dyad running parallel to z axis. Subsequent ultracentrifugation studies have demonstrated that PTE behaves as a dimer (Benning et al., 2000; 2001; Shim and Raushel, 2000; Raushel, 2002) (Fig. 2). The active site of phosphotriesterase is composed of a binuclear metal center of undefined structure that is required for catalysis. The three-dimensional structure of PTE was solved several years ago by X-ray Crystallographic analyses and an alpha carbon trace of subunit of homodimer. The protein folds into an (á â) 8 motif that is very similar in size and shape to urease (Jabri et al., 1995) and adenosine deaminase (Wilson et al., 1991). The active site is located adjacent to a binuclear metal center located with the aß-barrel. The native enzyme contains Zn 2+, but the divalent cations can be substituted with Cd2+, Co2+, Ni2+, or Mn2+ without loss of catalytic activity (Omburo et al., 1992). The two metal ions are co-ordinated to the protein via complexation with four histidine residues (His55, His57, His201, His230) and a single carboxilate from Asp301 (Benning et al.,1994; Kuo and Raushel, 1994). In addition to these legands, the two metal

is associated with high density lipoproteins (HDL). Not only distribution and structural features but also there is functional diversity of PTEs and the related OP hydrolyzing enzymes. In addition to PTEs many other related enzymes show PTE type activity. Esterase gene est-5S isolated from a rumen metagenomic library coded enzyme capable of degrading chlorpyrifos (CP) (Kambiranda et al., 2009). Eight other OPs were also tested for hydrolytic activity of the enzyme and many of them were more or less detoxified by the enzyme but at a comparatively slower rate (Dumas et al., 1989; 1990a, b; Huang et al., 2001; Kambiranda et al., 2009). Serum paraoxonases (PON1) found in mammals is an effective hydrolyser of paraoxon with its distribution in birds, frogs and fish. It is an enzyme of the gene family which also includes PON2 and PON3. These enzymes are lactonases and lactonising enzymes with not only overlapping, but also distinct substrate specificity (Draganov and La Du, 2004; Draganov et al., 2005). PON1, however, differs significantly in structure from that of bacterial PTEs. An Organophosphorus acid anhydrase (OPAA) has been isolated from bacteria by Cheng et al.(1999). The protein appears to be a proline dipeptidase and is able to catalyze the hydrolysis of OP chemicals. Similarly Liu et al. (2001) have purified a dimethoate degrading enzyme from Aspergillus niger ZHY256. The enzyme was found to be catalytically different from PTE as it does not catalyze the degradation of parathion. On the other hand, it degrades dimethoate, malathion and fumethion almost at the same rate. Esterases with OP degrading activity has also been reported in the crop Solanum melongena. Prolonged exposure of the plants to the OP insecticide dimethoate caused rapid esterase mediated hydrolysis of the insecticide (Mohapatra et al., 2010; Khillar et al., 2010). Similarly in Chlorella vulgaris exposure of the alga to dimethoate resulted in induction of tolerance but the mechanism of tolerance has not yet been determined (Jena et al., 2012). Structure of Pte The three dimensional structure of PTE was first developed by Benning et al. (1994). The molecule is roughly globular with overall dimensions of approximately 51Å×55 Å×51Å. Its molecular architecture may be described simply as a distorted á/â barrel and flanked on the outer surface 14 á helices. In addition to this major tertiary structural elements, there are two strands of anti parallel â sheets at the N-terminus, 13 type I turns, one type I’ turn, two type II’ turns and one helical turn. There are also a number of secondary structural elements as given in the table-1 with the â strands labeled as A-J. Two of â strands namely, C and E are disrupted by bulges forming by either His-55 (Ö=123º, Ø=-168º), respectively. The á helix formed by Gln 343 - Leu 358 is also irregular due to Val351 , which adopts dihedral angels of Ö=-121º and Ø=-58º (Dumas et al., 1989, 1990a, b; Benning et al., 1994, 1995, 2000, 2001; Raushel, 2002). There is not much similarity between PTE and other enzymatic proteins when primary structural homology is considered. However, the á/â barrel motif found in PTE have also been observed in well over 20 other enzyme (Aubert et al., 2004; Roodveldt and Tawfik, 2005). This constitutes most of the structure of PTEs, triose phosphate isomerase and many other esterases. Most of these enzymes have their active sites located in C- terminal portion of â barrel.

Figure 1: Some reactions catalyzed by known phosphotriesterases: DFPase, paraoxonase and HDCPase

4

PHARMACOLOGICAL EFFICACY OF MEDICINAL PLANTS

from Geobacillus caldoxylosilyticus might have been the ancestor of PTEs (Yildirim et al., 2009) but it differs from the later in having no phosphotriesterase activity. The PHPs are related to PTE in terms of amino acid sequence and the structure of bimetal catalytic site. This PHP is mutated to get extremely thermostability and pH stability and resistance to metal ions and organic solvent (Buchbinder et al., 1998; Raushel and Holden, 2000; Roodveldt and Tawfik, 2005; Yildirim et al., 2009). In E coli targeted mutation has caused directed evolution of PHP to show increased expression of PTE activity (McLoughlin et al., 2005). Sequence analysis revealed a presence of 921 base pair (bp) orf including a polypeptide of 307 amino acid residue corresponding to a molecular weight of 34.7 kd in PHP of Geobacillus caldoxylosilyticus (Yildirim et al., 2005). It was observed that in PTE the gene ranges in length from 855 bp to 963 bp encoding a protein of approximately 40kd. The residue coordinating the two catalyzed metals are entirely conserved. These Histidine residues (His55, His57, His201, and His230 of PTE) play the most important role in the catalytic activity of the enzyme (Lai et al., 1994; Benning et al., 1994). Homology analysis between the PHP and PTEs revealed that the amino acid residues of PHP corresponding to the His residue of PTE was found highly conserved (Fig. 3). In addition a ligating residue corresponding to Lys169 of PTE is also conserved but it is generally replaced by a Glu residue as seen in Geobacillus caldoxylosilyticus and an Ala residue in E. coli (Buchbinder et al., 1998; Raushel and Holden, 2000; Yildirim et al., 2005, 2009). A comparison of PTE of Psudomonas diminuta (PDPTE), with PHP of Geobacillus caldoxylosilyticus (GCPHP), Micobacterium tuberculosis (MTPHP) and E. coli (EPHP) showed that the loops of first seven and eight á â modules of PTE differ from that of PHP (Fig. 3). These modules are most upon involved in contacting the ligands in the active site and determining the substrate specificity of the enzymes (Seibert and Raushel, 2005). These modules are sorter in PHP of Geobacillus caldoxylosilyticus and E. coli than in PTE of Psudomonas diminuta and Flavobacterium spp. indicating that the lack of catalytic activity of PHP is due to their inability to recognize the substrate (Yildirim et al., 2009). The PTE of the other sources like of Sulfolobus acidocaldarius (SAPTE) and Sulfolobus sulfataricus (SSPTE) also showed identical homology with PDPTE as well as with PHP (GCPHP, EPHP and MTPHP) (Porzio et al., 2007). Phylogenetic analysis of selected PTE (PTE of Psudomonas diminuta; Flavobacterium spp. ATTCC 27551 and Flavobacterium MTCC 2495), PHP (PHP of Geobacillus caldoxylosilyticus TK4 and E. coli) and Phosphotriesterase related protein (PRP) (PRP of Homo sapiens, Rattus norvegicus and Mus musculus) showed that the PRP of Homo sapiens is found to be quite distinct of homologous proteins (Fig. 4). Maximum homology was observed between the PTE of two Flavobacterium spp. and they are also found to be closely related with the PTE of Psudomonas diminuta. The PHP of Micobacterium tuberculosis was found to be the closest relative of PTE of three bacterial species (Porzio et al., 2007; Yildirim et al., 2009). In addition to PTE a number of microorganisms cause degradation of OP pesticides by synthesis of a quite unrelated enzyme. A strain of Psudomonas sp. WBC3 was found to synthesize an enzyme called methyl parathion hydrolase (MPH) on induction with methyl

A

B

C

Figure 2: The structure of DFPase: (a) A line drawing showing the Ctrace with every tenth residue labeled and the position of two Ca2+ ions (black dots) ; (b) The overall structure viewed down the pseudo six-fold axis (1 – 6), each consisting of four strands A–D; (c) A side view of the enzyme

ions are bridged via a carboxilated lysine residue and a hydroxide from a solvent (Lai et al., 1994, 1995; Benning et al., 1995). The bridging hydroxide acts as a nucleophile for the hydrolytic attack on the phosphorus center of the substrate (Lai et al., 1995; Benning et al., 2000; Hawwa et al., 2009). Structural Homology of Pte The study of the homology of PTEs of bacteria, fungi, mammals, fish and birds as well as the homology of PTEs with other related proteins is an area of importance in the recent times. PTE has been found to be a fine conserved enzyme though it is distributed in many of the organisms more especially in microbes. The number of amino acid residues range from 280-365 thus indicating a variation of about 15% in its primary structure. Phylogenetic analysis based on amino acid sequence shows that the PTEs of different organisms are most closely related to the PHP of Geobacillus caldoxylosilyticus and E. coli (Lai et al., 1995; Yildirim et al., 2005, 2009; Hess et al., 2008). The closest sequence homologue identified to date is PHP, an enzyme with unknown function. PHP is a member of amidohydrolase superfamily and exhibits 28% sequence identity, and 66% sequence similarity to PTE (Roodveldt and Tawfik, 2005). Both enzymes possess an essentially identical binuclear (Zn2+) metal center (Lai et al., 1994; Roodveldt and Tawfik, 2005). PHP 5

P. K. MOHAPATRA AND SUCHISMITA PATTANAIK E HP HP

M -

Q T - - - - - -

R R V V - - - - - - - - - - - - - - - -

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E E HP HP E E HP HP E E HP HP E E HP HP E E HP HP E

F V V Y F F

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T L T H E T L M H E C A C H E T L A H E T L I H E T L I H E V L M M P P

A A A A G T - - - - - - - - - - - - - - - - - - - - - - - - - -

L -

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G G L A G C A - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

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R A G I I K - V A T T G K A G I L K - C A T D E P G V I G E I G S S F N K A G I I A E I G T S E G K A G F V K - I A A D E R A G F I K - V A A D E -

174 151 140 131 143 144

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E S E A E E I R E Q A H E E G E E G

219 196 184 175 190 191

E D N A - - - - - - - - - - - - - - - -

266 238 226 217 230 231

Loop 7

L M L F L L

D D D D D D

H I P H S A R F G V D V T I G K E N T I G K N S R Y G L D L R Y G L D L

I G L I S Y R Y Y F - F - -

S S Y V T D A L P E - - - - - - - - D - W G T D - W G I

S S S S S S

N H S M H Q

D D D D D D

W A V I Y Y

K H M S N A

E E T A E E

T Q T D V Q

L L I V +I L

A H E D A H

G I T T M L K L L V M L T I F V I F

V P Q V T D V L E F S Q V N E V T D

G G G - K K G

R A E Q N N

V L L L I V

G G G G G G

R R L G G

V I V C V V

M K Q I L K M V E D Q I L L N R V M S D K I M L D R I M

+ A * D

V A V V V

L C T T C C

F C R R C C

Loop 8 G Y T T

F

F + I I

V T N P A R F V D N P R R I V K N P Q K A R E N P S Q F K E N P K K F V K N P A R L

P T L R A S R Q G G Y Q I R K E G - - - - - - - - - - - - - - - -

N E A A

I L K K

313 276 255 246 268 269

L S F E F S F Q F S F S

359 320 301 292 315 316 365 326 306 292 315 316

+

*

Please send JPG fomat for source file

+

++

++

Figure 3: Structural multisequence alignment among PTE of Pseudomonas diminuta (PDPTE; Buchbinder et al., 1998), Sulfolobus sulfataricus (SSPTE; Porzio et al., 2007), Sulfolobus acidocaldarius (SAPTE; Porzio et al., 2007) and PHP of Mycobacterium tuberculosis (MTPHP; Yildirim et al., 2009), Geobacillus caldoxylosilyticus (GCPHP; Yildirim et al., 2009) and Escherichia coli (EPHP; Buchbinder et al., 1998).The loops of the first, seventh and eighth â/á modules have been shown in bold letters.conserved residues from the conserved cluster of the binuclear metal centre (His55, His57, His201, His230 and Asp301 of PDPTE) are marked with an asterisk. The other conserved residues are marked with a plus

Flavobacterium spp. (Mulbry and Karns, 1989), respectively. Thus like PTE, MPH was found to a member of metallo â lactamase family with significant conservation of metal coordinating residues in the binuclear metal center. Despite the lack of any sequence or structural homology with PTE, there are obvious similarities in the metal binding centers that suggest a common catalytic mechanism. Jackson et al. (2008) reported a PTE, structurally and functionally similar to

parathion. The bacterium is able to use methyl parathion as a sole C/N source to grow and can completely degrade the insecticide to P-nitrophenol and phosphate (Dong et al., 2005). The mpd gene encoding MPH from Psudomonas sp. WBC3 shows 93% and 92% sequence homology with Psudomonas putida and Plesiomonas sp. M6 respectively. However, mpd lacks the sequence homology with the opd gene including PTE from Psudomonas putida (Dumas et al., 1989) and 6

There is a proven fact that PTE has the ability to hydrolyze most of the OPs making it a strong candidate for application in their detoxification. Due to these properties PTE has been described as a potential catalytic scavenger of treatment of OP poisoning (Masson et al., 1998; Li et al., 2000). There is potentiality of using PTE as a degrading enzyme either in liquid suspension or as immobilized surface for treatment of water pollution by OP pesticides. Proper concentration of the enzyme as well as its mode and medium of administration are required to be determined for fast and efficient detoxification of OP chemicals. However, the major bottle neck in this area is the broad range of structural variation of OP chemicals, which sometimes are not properly recognized by the enzyme. For example Istamboulie et al. (2010) observed that addition of a mixture of chlorpyrifos (CP) and chlorfenvinfos (CFV) to a medium containing PTE causes rapid degradation of CP but slow metabolism of CFV indicating the limitation of the enzyme to recognize both the substrate with same efficiency. CFV rather acted as a competitive inhibitor with a very slow rate of hydrolysis at a comparatively high concentration of the enzyme. Thus there is a need to work on the design of the enzyme to recognize a broad range of OPs. Another aspect to be taken care of in the research on development and application of PTEs is the maintenance of the catalytic activity of the enzyme in the cell free medium and carrier surfaces. Lyophilization has been used as a means to minimize water surrounding the enzyme which limit protein denaturation and proteolysis during storage (Le-Jeune and Russel, 1996; Sogorb et al., 2004). Lyophilized enzyme in a continuous gas phase reactor was remarkably active in hydrolysis of paraoxon and lyophilized enzyme introduced into polyurethene foam retained 52% of the initial specific activity from the enzyme preparation. This requires a further improvement of the procedure to enhance the catalytic activity during therapeutic application. Immobilization of partially purified enzymes in pore glass and silica beads are more effective in maintaining the catalytic activity. The studies have demonstrated that the

Figure 4: Phylogenetic analysis of the selected phosphotriesterases, phosphotriesterase homology proteins and phosphotriesterase-related proteins constructed by comparison of deduced amino acid sequences of G. caldoxylosilyticus TK4PHP (TK4PHP), P. diminuta PTE (P0A434), Flavobacterium sp. (strain ATCC 27551) parathion hydrolase (P0A433), Flavobacterium sp. MTCC 2495 organophosphorus hydrolase (Q5UB52), Homo sapiens phosphotriesterase-related protein (Q96BW5), Rattus norvegicus phosphotriesterase-related protein (Q63530), Mus musculus phosphotriesterase-related protein (A2AUR4), E. coli PHP (P45548) and M. tuberculosis PHP (P96413) with the maximum parsimony method (Yildirim et al., 2009)

Psudomonas diminuta, from Agrobacterium radiobacter. The enzyme identified from the extremophile Dinococcus radioduarns was found to have 48% sequence homology with PTE and has been shown to have OP degrading ability (Hawwa et al., 2009; Xiang et al., 2009). However, Hawwa et al. (2009) observed that the native enzyme show low OP degrading ability and the engineered OP and site directed mutagenesis could promote the catalytic activity of the enzyme. Future Prospects Table 1: List of Secondary structural elements of PTE Amino acid residues Type of structure Arg 36 - Asn 38 Thr 39 - Gly 42 Pro 43 - Thr 45 Ile 46 - Ala 49 Phe 51 - Glu 56 Gly 60 – Ala63 Ser 62 – Phe 65 Leu 66 – Ala 68 Pro70 – Phe 73 Arg 76 – Ala93 Thr 97 – Asp 100 Phe 104 – Gly 107 Val 110 – Ser117 Arg 118 – Asp 121 Val 122 – Trp 131 Leu 136 – Met 138 Met 138 – Arg 141 Val 143 – Gln 155 Ile 158 – Thr 161 Ile 167 – Thr 173 Pro 178 – Thr 194

²-Sheet (A) Type I turn ² – Sheet (B) ± –Helix ² – Sheet (C) type I turn type I’ turn Helical turn ± –Helix ± –Helix ² – Sheet (D) Type I turn ± –Helix Type I turn ² – Sheet (E) ± –Helix Type I turn ± –Helix Type II’ turn ² – Sheet (F) ± –Helix

Amino acid residues

Type of structure

Pro 197 – his 201 Asp 208 – phe 216 Glu 217 – Gly 220 Ser 222 – Arg 225 Val 226 – His 230 Leu 237 – Leu 243 Ala 244 – Gly 247 Leu 249 – Leu 252 Trp 277 – Leu 287 Ile 288 – Gly 291 Tyr 292 – Gln 295 Ile 296 – Ser 299 Asn 300 – Phe 304 Ser 308 – Val 310 Thr 311 – Met 314 Asn 321 – Gly 324 Asp 323 – Ala 326 Phe 327 – Leu 336 Arg 337 – Gly 340 Gln 343 – Leu 358

²-Sheet (G) ± –Helix Ü type I turn Ü type I turn ² – Sheet (H) ± –Helix type I turn ² – Sheet (I) ± –Helix type I turn type I turn ² – Sheet (J) ± –Helix ± –Helix type I turn type I turn type II’ turn ± –Helix type I turn ± –Helix

7

294.

immobilized enzyme accomplished 95% hydrolysis of up to 250 μg/mL parathion with no loss of activity during 70 days of continuous flow experiment but problems lie with the extraction of the entrapped enzyme from the immobile surface (Di Sioudi et al., 1999). Enzymes concentrated in columns or encapsulated within sterically stabilized liposomes are found effective in maintaining their stability and catalytic activity (Petrikovics et al., 1999; Ghanem and Raushel, 2005; Andreescu and Marty, 2006; Istamboulie et al., 2007, 2010). There is a need to develop surface that can not only optimize the catalytic activity but also maximize the release of enzyme from the surface more effectively. More recently researches are being done to address the issue of enzyme stability under extreme conditions (stressed with temperature, salt and pH). Enzymes isolated from thermophilic microbes are being tried to treat OP poisoning under a variety of field conditions (Grunden et al., 2004; Theriot et al., 2010a; Theriot and Grunden, 2011).

Aubert, S. D., Li, Y. and Raushel, F. M. 2004. Mechanism for the hydrolysis of organophosphates by the bacterial phosphotriesterase. Biochemistry. 43: 5707-5715. Baek, J. H., Clark, J. M. and Lee, S. H. 2010. Cross-strain comparison of cypermethrin-induced cytochrome P450 transcription under different induction conditions in diamondback moth. Pestic. Biochem. Physiol. 96: 43–50. Benning, M. M., Kuo, J. M., Raushel, F. M. and Holden, H. M. 1994. Three-dimentional structure of phosphotriesterase: An enzyme capable of detoxifying organophosphate nerve agents. Biochemistry. 33: 1500115007. Benning, M. M., Kuo, J. M., Raushel, F. M. and Holden, H. M. 1995. Three-dimensional structure of the binuclear metal center of phosphotriesterase. Biochemistry. 34: 7973-7978. Benning, M. M., Hong, S. B., Raushel, F. M. and Holden, H. M. 2000. The binding of substrate analogs to phosphotriesterase. J. Biol. Chem. 275: 30556-30560. Benning, M. M., Shim, H., Raushel, F. M. and Holden, H. M. 2001. High resolution X-ray structure of different metal – substituted froms of phosphotriesterase from Pseudomonas diminuta. Biochemistry. 40: 2712 – 2722.

CONCLUSION Hydrolysis plays the central role in the detoxification of OPs. Mutant forms of several bacterial PTEs have been created in the last years with an aim to enhance the hydrolyzing activity towards substrates that are hydrolyzed by PTEs with poor effectiveness. Mutants of PTE found in Pseudomonas diminuta have been constructed with substrate profiles that are optimized with the hydrolysis of chemical warfare agents and insecticides. The study on the enzyme and its catalytic activity will have an impact on the design of more efficient and at the same time safer insecticides. The study of structure-activity relationship seem also important, as it will allow to design compounds susceptible to hydrolysis by the PTEs and therefore, easily degraded by non-target species. Understanding such a relationship might also important in case of the hydrolysis of OPs associated to albumin. The protein is universally present in the serum of all vertebrates and its hydrolyzing activity might be especially important in individuals, or in species, laking other types of hydrolyzing enzymes. All available data allow expecting that it will shortly be possible to design PTE mutants as specific antidotes for specific OPs. There are sufficient experimental data to suggest that it is now possible to initiate clinical trials with PTEs. Such trials needs international collaborations focusing the use of PTEs within biological carriers (erythrocytes and liposomes) to apart the classical pharmacological treatments. A systematic study on the substrate-enzyme interaction will make it possible to create conditions for minimization of non-target toxicity of OP chemicals.

Buchbinder, J. L., Stephenson, R. C., Dresser, M. J., Pitera, J. W., Scanlan, T. S. and Fletterick, R. J. 1998. Biochemical characterization and crystallographic structure of an Escherichia coli protein from the phosphotriesterase gene family. Biochemistry. 37: 5096–106. Cheng, T. C., Harvey, S. P. and Stroup, A. N. 1993. Purification and properties of a highly active organophosphorus acid anhydrolase from Alteromonas undina. Appl. Environ. Microbiol. 59: 3138-3140. Cheng, T. C., DeFrank, J. J. and Rastogi, V. K. 1999. Alteromonas prolidase for organophosphorus G-agent decontamination. Chem. Biol. Interact. 120: 455-462. Cheng, Y. D., Karns, J. S. and Torrents, A. 1998. Characterization of a phosphotriesterase from genetically engineered Escherichis coli. J. Environ. Sci. Health. B33: 347 – 367. Corbett, J. R. 1974. The Biochemical Mode of Action of Pesticides. Academic Press, New York. Di Sioudi, B. D., Miller, C. E., Lai, K. H., Grimsley, J. K. and Wild, J. R. 1999. Rational design of organophosphorus hydrolase for altered substrate specificities. Chem. Biol. Interact. 120: 211-223. Dong, Y. J., Martlam, M., Sun, L., Zhou, Y. F., Zhang, Z. P., Zhang, C. G., Rao, Z. and Zhang, X. E. 2005. Crystal structure of methyl parathion hydrolase from Pseudomonas sp. WBC-3. J. Molec. Biol. 353: 655-663. Draganov, D. I. and La Du, B. N. 2004. Pharmacogenetics of paraoxonases: a brief review. Naunyn Schmiedebergs Arch. Pharmacol. 369: 78–88. Draganov, D. I., Teiber, J. F., Speelman, A., Osawa, Y., Sunahara, R. and La Du, B. N. 2005. Human paraoxonases (PON1, PON2, and PON3) are lactonases with overlapping and distinct substrate specificities. J. Lipid Res. 46: 1239–1247. Dumas, D. P., Caldwell, S. R., Wild, J. R. and Raushel, F. M. 1989. Purification and properties of the phosphotriesterase from Pseudomonas diminuta. J. Biol. Chem. 264: 19659–19665.

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