Open Access

Marked enhancement of Acinetobacter sp. organophosphorus hydrolase activity by a single residue substitution Ile211Ala

Bioresources and Bioprocessing20152:39

DOI: 10.1186/s40643-015-0067-3

Received: 22 June 2015

Accepted: 25 August 2015

Published: 15 September 2015



The activity of organophosphorus hydrolase (OPH) that catalyzes the hydrolysis of neurotoxic organophosphates (OPs) was reported to evolve from lactonase.


In this study, a putative OPH from Acinetobacter sp. (AbOPH) exhibited high lactonase activity with latent OPH activity. Sequence alignment and phylogenetic tree analysis revealed the unique status of AbOPH in evolution. The crystal structure of AbOPH was determined at 2.0 Å resolution and a semi-rational design was performed to enhance the OPH activity of AbOPH through a consensus sequence approach. Compared with wild-type AbOPH, which exhibited undetectable activity toward methyl-parathion (MP), the best variant AbOPHI211A showed markedly improved catalytic efficiency (1.1 μmol min−1 mg protein −1 ) toward MP. Docking studies suggested that the mutation Ile211Ala affects substrate recognition and stabilizes substrate conformation.


This result presents the emergence of new enzyme function by a simple mutation strategy and confirms the high possibility that OPH was evolved from its lactonase ancestor.


Crystal structure Organophosphorus hydrolase Lactonase Semi-rational design Site-directed mutagenesis


Organophosphates (OPs) are common neurotoxic compounds (Singh 2008) that have been extensively used as agricultural insecticides (Raushel 2002). Massive use of these pesticides has brought serious threats to environmental safety and human health (Sapozhnikova et al. 2004, 2005). Enzymatic degradation of OPs was considered as a desirable decontamination method with many advantages, such as being economic, efficient and environmentally friendly (Singh and Walker 2006). A number of enzymes capable of degrading OPs have been discovered during the past years, including organophosphorus acid anhydrolases (OPAAs; EC (Cheng et al. 1999; Vyas et al. 2010), phosphotriesterases (PTEs; EC (Omburo et al. 1992), paraoxonases (PONs; EC (Ben-David et al. 2012), phosphotriesterase-like lactonases (PLLs; EC (Afriat et al. 2006), and methyl-parathion hydrolases (MPHs; EC (Hong et al. 2005).

In recent years, clarifying the evolutionary pathway (Bar-Rogovsky et al. 2013; Luo et al. 2014) and structure–activity relationship (Dong et al. 2005; Gotthard et al. 2013) of organophosphorus hydrolase (OPH) has been extensively studied. Phosphotriesterase (PTE) from Brevundimonas diminuta was the best-characterized and potentially important OPH, which exhibited near diffusion-limit reaction rate (107–108 M−1 s−1) to ethyl-paraoxon (Omburo et al. 1992). PTE was reported to emerge from native lactonases (PLLs) with promiscuous OPH activity (Merone et al. 2010; Elias et al. 2008; Hawwa et al. 2009). In addition, mammalian paraoxonase (PON) also showed native lactonase activity (Khersonsky and Tawfik 2005), which was believed to evolve from mammalian paraoxonase ancestor with bifunctionality (HSLase and δ-/γ-lactonase) (Bar-Rogovsky et al. 2013).

The MPH isolated from Pseudomonas sp. strain WBC-3 catalyzes the degradation of the organophosphate pesticide methyl-parathion (MP) with a high efficiency (k cat/K M ~106 M−1 s−1) (Dong et al. 2005). The structure of MPH (PDB ID: 1P9E) has been resolved, revealing a typical αβ/βα sandwich fold structure of metallo-β-lactamase superfamily (Dong et al. 2005). In parallel with MPH, a newly identified OPH, OPHC2, isolated from Pseudomonas pseudoalcaligenes, is a thermostable OPH (T m = 97.8 ± 3.2 °C) (Gotthard et al. 2013) in β-lactamase superfamily, which shares 46 % sequence identity with MPH. PoOPH, a newly discovered lactonase from Pseudomonas oleovorans, exhibited high lactonase and esterase activities, but low OPH activity (Luo et al. 2014). PoOPH shares 98.5 % sequence identity with OPHC2. By simple double mutations His250Ile/Ile263Trp, PoOPH was switched into an efficient OPH, displaying 6962-fold improvement in catalytic efficiency toward MP (Luo et al. 2014). The study proves the emergence of efficient and robust enzymes for OP detoxification by OPH activity evolution in the β-lactamase superfamily.

In this study, a distinctive putative OPH (AbOPH) identified from Acinetobacter sp. exhibited high lactonase activity with latent OPH activity. Compared with OPHC2 and MPH in β-lactamase superfamily, AbOPH acts as a separate branch in the evolutionary tree and there are four additional amino acids (Gly206, Thr207, Val208 and Glu209) in its sequence. A semi-rational design was performed for AbOPH based on its crystal structure and sequence analysis to find the key residues that affect its OPH activity. The best variant AbOPHI211A was characterized and the possible mechanism for the improvement of OPH activity was investigated by molecular docking studies.


Chemicals and strains

OPs were purchased from Shanghai Pesticide Research Center. para-Nitrophenyl butyrate (pNPB) was synthesized in our laboratory. 3,4-Dihydrocoumarin, δ-nonanolactone and γ-decanolactone were obtained from TCI Co., Ltd. (Tokyo, Japan). All chemicals used for crystallization were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Other chemicals with reagent grade or better quality were obtained from commercial sources. Escherichia coli (E. coli) DH5α and E. coli BL21(DE3) (Novagen, Germany) were used for gene amplification and expression, respectively (Du et al. 2014). Acinetobacter sp. (NBRC 100985) used in this study was purchased from NBRC (NITE Biological Resource Center).

AbOPH cloning and site-directed mutagenesis

AbOPH gene was amplified by PCR using whole genome of Acinetobacter sp. as template with the following primers: 5′-GGGTTTCATATGATGCTAAAAAATAGAC-3′ (forward) and 5′-CCCAAGCTTATCTTTAAAATGGATCGGA-3′ (reverse) (Luo et al. 2014). The PCR fragment was subsequently cloned into expression vector pET-28a(+) to generate the recombinant plasmid pET28a–AbOPH. Site-directed mutations of AbOPH were constructed using QuickChange® Site-Directed Mutagenesis Kit (Stratagene, USA), and the plasmids carrying the gene of AbOPH and the desired mutations were transformed into E. coli DH5α for amplification and then transformed into E. coli BL21(DE3) cells for expression.

Protein expression and purification

A single colony of recombinant strain was cultivated in LB medium containing 50 μg/mL kanamycin at 37 °C. When OD600 reached 0.6–0.8, the cultivation temperature was decreased to 16 °C and isopropyl β-d-thiogalactopyranoside (IPTG) was added with a final concentration of 0.5 mM to induce protein expression. After 20 h, the cells from 1 L culture were collected by centrifugation (8800×g) at 16 °C, resuspended in buffer A (20 mM Tris–HCl, 500 mM NaCl, 20 mM imidazole, pH 8.0) and lysed by French Press. The cell lysate was centrifuged at 30,000×g, 4 °C for 1 h; then the supernatant was loaded onto a Ni2+-NTA affinity column (5 mL, GE Healthcare Co.). After prewashing with buffer A, the recombinant protein was eluted with buffer A containing imidazole with linear gradient from 10 to 500 mM. The target protein collected was then loaded onto a HiLoad 16/60 Superdex 75 preparative column (120 mL, GE Healthcare Co.) and eluted by Tris–HCl buffer (25 mM, pH 7.5) containing 150 mM NaCl and 1 mM dithiothreitol for protein condensation. SDS-PAGE analysis of the eluted protein revealed over 90 % purity of the target protein. The final volume and concentration of the concentrated AbOPH protein were 1 mL and 14 mg/mL, respectively.

Crystallization, data collection and structure refinement

Crystallization of AbOPH was carried out at 4 °C using sitting-drop vapor diffusion method with crystallization buffer containing 0.02 M d-glucose, 0.02 M d-mannose, 0.02 M d-galactose, 0.02 M l-fucose, 0.02 M d-xylose, 0.02 M N-acetyl-d-glucosamine, MES/Imidazole (0.1 M, pH 6.5), 12.5 % (w/v) PEG1000, 12.5 % (w/v) PEG3350 and 12.5 % (v/v) 2-methyl-2,4-pentanediol. The protein concentration for crystallization was 14 mg/mL. Crystals that appeared were transferred to cryoprotectant solution composed of mother liquors supplemented with 5 % (v/v) ethylene glycol prior to flash cooling in liquid nitrogen. X-ray diffraction data were collected with flash-frozen crystals (at 100 K in a stream of nitrogen gas) on a RaxisIV++ imaging plate (Rigaku, TX, USA) using an in-house Rigaku MicroMax-007 HF rotating-anode X-ray generator operating at 40 kV and 30 mA. The intensity sets were indexed, integrated and scaled with the HKL2000 package (Otwinowski and Minor 1997). The initial structure of AbOPH was determined by molecular replacement with PHASER (McCoy et al. 2007) using OPHC2 (PDBID: 4LE6) as the template. Several cycles of refinement were carried out using Phenix and Coot (Murshudov et al. 1997; Emsley and Cowtan 2004).

Activity assay and kinetic parameters determination

The enzyme activities toward various OPs (methyl-parathion, ethyl-paraoxon, malathion, fenitrothion, dimethoate and diazinon), lactones (3,4-dihydrocoumarin, δ-decanolactone and γ-nonanolactone) and esters (2-naphthyl acetate and para-nitrophenyl butyrate) were determined as described (Gotthard et al. 2013; Khersonsky and Tawfik 2005; Dumas et al. 1989; Zhang et al. 2012; Roodveldt and Tawfik 2005). The kinetic parameters were determined by measuring activities toward MP at concentrations from 0.01 to 1 mM and fitting data to the Michaelis–Menten equation.

Homology modeling and molecular docking

The structure of AbOPHI211A was modeled using AbOPH crystal structure as the template on SWISS-MODEL sever ( The substrate MP was docked into AbOPHI211A and AbOPH, respectively, using Discovery Studio 2.5 software. Two zinc ions were selected as binding site. The radius of the SBD site sphere was set as 9 Å. LibDock protocol was used in further docking simulation. The conformations from the calculation that conflict with the catalytic mechanism were initially ruled out. The best-scoring conformation was then subjected to detailed analysis.

Results and discussion

Identification and characterization of a putative OPH

To identify new enzymes with potential OPH activities, OPHC2 was chosen as a probe to search for homolog proteins based on BLAST (Basic Local Alignment Search Tool) Web server in the NCBI (National Center for Biotechnology Information) database. Eleven OPHC2-like proteins with sequence identity ranging from 40 to 98 % were selected as candidates (Fig. 1a, Additional file 1: Figure S1). All of these proteins belong to β-lactamase superfamily and show entirely conserved metal ion coordination residues. From the evolutionary tree analysis, two putative OPHs (AbOPH and SmOPH) act as separate branches, belonging to neither MPH nor OPHC2 clade (Fig. 1b) (Luo et al. 2014). Based on the results of sequence alignment, the AbOPH seems quite special since there are four additional amino acids (Gly206, Thr207, Val208 and Glu209) in its sequence, which do not exist in other putative OPHs. The putative OPH from Acinetobacter sp., hereafter designated as AbOPH, shares merely 44 % sequence identity with OPHC2 and 38 % with MPH. The AbOPH was heterologously overexpressed in E. coli, purified and characterized. The molecular weight of purified AbOPH was about 37 kDa according to SDS-PAGE analysis (Additional file 1: Figure S2A). Similar to PoOPH (Luo et al. 2014), AbOPH also exhibited some functional promiscuity, with high lactonase activity (996 μmol min−1 mg−1 toward 3,4-dihydrocoumarin) and latent OPH activity (only 0.04 μmol min−1 mg−1 toward fenitrothion and no activity was detected toward MP).
Fig. 1

Phylogenetic analysis of AbOPH. a Multiple sequence alignment of AbOPH (ZP_09219954) with enzymes in β-lactamase superfamily. Sequences were selected from BLAST in the NCBI (sequence identity >40 %) using input query of OPHC2 from Pseudomonas pseudoalcaligenes (CAE53631), including SmOPH from Stenotrophomonas maltophilia (CCH14104), PoOPH from Pseudomonas oleovorans (4O98_A), PpOPH from Pseudomonas putida (AAR21562), YrOPH from Yersinia rohdei (ZP_04610908), HgOPH from Hylemonella gracilis (ZP_08405683), DpOPH from Desulfobulbus propionicus (YP_004194377), DdOPH from Dickeya dadantii (YP_003884376), PcOPH1 and PcOPH2 from Pectobacterium carotovorum (ZP_03833759; ZP_03833761), MPH from Pseudomonas sp. strain WBC-3 (AAP06948) and VpOPH from Variovorax paradoxus (YP_002945503). Secondary structures are annotated according to the AbOPH structure. Additional residues 206–209 are highlighted by a blue box. Ile211 is indicated with red arrow. Alignment was performed using MUSCLE server and displayed using Esprit ( b Phylogenetic tree analysis of enzymes in β-lactamase superfamily. Phylogenetic tree was built using Discovery Studio 2.5 software. Two major clades are shown in the tree: MPH clade and OPHC2 clade

Crystal structure of AbOPH

The crystal structure of AbOPH was solved at 2.0 Å resolution (PDB ID: 4XUK) (Additional file 1: Figure S2B). The crystal contains two molecules in the asymmetric unit and belongs to the space group P212121 with unit cell parameters a = 76.8, b = 82.7, c = 99.8 and α = β = γ = 90°. Refinement of the structure finally converged with an R-work of 0.204 and R-free of 0.238. The final model comprised 291 amino acids, 2 zinc ions and 242 water molecules. Because the residues 1–47 were absent in the electron density maps, they were not modeled. The monomer of AbOPH is roughly ellipsoidal with approximate dimensions of 52 Å × 50 Å × 32 Å. Data collection and refinement statistics are summarized in Table 1.
Table 1

Data collection and refinement statistics



Data collection statistics


Rigaku Micromax-007 HF&Raxis IV++

 Space group

P21 21 21

 Unit cell (Å/deg.)

a = 76.8, b = 82.7, c = 99.8, α = β = γ = 90.0

 Wavelength (Å)


 Resolution range (Å)a

50.00–2.00 (2.05–2.00)

 Total/unique reflections



12.0 (11.6)

 Completeness (%)a

98.6 (99.1)

 Mean I/σ(I)a

31.3 (8.7)

 Wilson B factor (Å2)


 Solvent content (%)


Refinement statistics

 Resolution range (Å)a

34.83–2.00 (2.05–2.00)

 R work (%)b


 R free (%)b


R.m.s. deviation

 Bond lengths (Å)


 Bond angles (deg.)


 Average B factor (Å2)


Ramachandran plot

 Favored (%)


 Disallowed (%)


aNumbers in parentheses are values for the highest-resolution shell

b R work = ∑||F oF c||/acF o|. R free = ∑T||F oF c||/eT|F o|, where T is a test data set of 5 % of the total reflections randomly chosen and set aside prior to refinement

AbOPH forms a homodimer in the asymmetric unit (Fig. 2a) and the structure could be described as αβ/βα sandwich topology (Fig. 2b), analogous to other members of β-lactamase superfamily (Luo et al. 2014; Gotthard et al. 2013). Each asymmetric unit is composed of a β-lactamase-like domain and the four additional residues 206–209 are located in helix α5. The binuclear metal center locates between the inner β-sheets of the αβ/βα sandwich. The two zinc ions are separated by a distance of 3.2 Å. The buried α zinc ion is coordinated by Asp159, His160, Asp265 and His312 and the less buried β zinc ion is coordinated by His244, His155, His157, Asp265 and a water molecule. Both metals are bridged by a putative catalytic water molecule (Fig. 2c).
Fig. 2

Crystal structure of AbOPH. a General representation of the AbOPH dimer. The two metals of the active site are represented as gray spheres. b View of overall structure of the AbOPH monomer. α helices are colored in blue, β sheets in magenta and loops in wheat color. The bimetallic center is shown as two gray spheres. Additional residues 206–209 are highlighted in red. c Active site of AbOPH. Residues are shown as sticks. The two metals are represented as gray balls and the two water molecules are shown as red spheres. The metal coordination sphere is shown as dashed line

Structural comparison of AbOPH with other OPHs showed that the overall conformation and the active center of AbOPH were similar to OPHC2 and MPH (Additional file 1: Figure S3), while the OPH activity of AbOPH was considerably lower than that of OPHC2 and MPH. Considering the high conformation similarities in contrast to huge differences of OPH activity between AbOPH, OPHC2 and MPH, a semi-rational design based on consensus sequence approach was performed to identify the key residues that depress the OPH activity of AbOPH.

Site-directed mutagenesis of AbOPH

Nineteen conserved residues of AbOPH (Additional file 1: Figure S4) within 10 Å from the active center were mutated into the corresponding amino acid residues of OPHC2 or MPH, respectively. The activities of these mutants were measured using MP as substrate. Only three variants, AbOPHI211A, AbOPHL156M and AbOPHH268L, exhibited detectable OPH activities (Table 2), suggesting that residues 211, 156 and 268 are crucial to OPH activity of AbOPH. Mutation H268L of AbOPH just corresponds to the H250L of PoOPH that confirms the previously public conclusions (Luo et al. 2014). The key residues 211 and 156 that determine OPH activity were identified for the first time, especially the variant AbOPHI211A showed the most improved OPH activity (Table 2). Saturation mutagenesis at Ile211 and combination mutagenesis Ile211/L156M/H268L were performed subsequently to further improve OPH activity; however, all the other variants exhibited extremely lower OPH activities than AbOPHI211A (Additional file 1: Figure S5).
Table 2

Specific activities and kinetic parameters of wild-type AbOPH and its variants


Specific activity (U/mg)

K M (μM)

k cat (min−1)

k cat/K M (min−1 μM−1)






1.060 ± 0.023

103 ± 14

93.2 ± 3.2



0.0115 ± 0.0010

45.5 ± 5.6

0.862 ± 0.031



0.0104 ± 0.0011

56.7 ± 7.0

0.804 ± 0.030


Specific activities of purified enzymes were measured toward 0.5 mM MP

The kinetic parameters were determined by measuring purified enzyme activities toward MP at concentration from 0.01 to 1 mM and fitting data to the Michaelis–Menten equation. All enzymatic assays were performed at least in triplicate and average values were adopted

ND no enzymatic activity was detected except spontaneous hydrolysis, in spite of the high enzyme loading (2 mg/mL)

The kinetic parameters of variants AbOPHI211A, AbOPHL156M and AbOPHH268L were determined by non-linear fitting. As shown in Table 2, the K M values of these three mutants were similar, while the k cat of AbOPHI211A was significantly higher than those of AbOPHL156M and AbOPHH268L. The catalytic efficiency (k cat/K M) of AbOPHI211A toward MP reached 0.9 min−1 μM−1, which was nearly 50 times higher than those of AbOPHL156M and AbOPHH268L. The higher k cat of AbOPHI211A was considered as the main advantage for enzymatic hydrolysis of MP.

Activities of AbOPHI211A toward several OPs, lactones and esters were determined (Table 3; Additional file 1: Figure S6). Compared with wild-type AbOPH, the OPH activity of AbOPHI211A was significantly enhanced and the lactonase activity was decreased, while the esterase activity was similar. AbOPHI211A showed significant activities toward MP and ethyl-paraoxon, while the activities of wild-type AbOPH toward those substrates were not detected. The activity of AbOPHI211A toward fenitrothion reached 0.54 μmol min−1 mg−1, 11-fold higher than that of AbOPH. Compared with AbOPH, AbOPHI211A exhibited decreased lactonase activities toward 3,4-dihydrocoumarin, δ-decanolactone and γ-nonanolactone, being 60, 93 and 89 %, respectively. Therefore, AbOPHI211A exhibited a trade-off between its activities of lactonase and OPH in the evolutionary process.
Table 3

Activities of AbOPH and AbOPHI211A toward various substrates


AbOPH (μmol min−1 mg−1)

AbOPHI211A (μmol min−1 mg−1)




1.68 ± 0.08



0.072 ± 0.012


0.048 ± 0.011

0.543 ± 0.045












996 ± 22

389 ± 10


54.1 ± 6.1

3.74 ± 0.56


14.8 ± 2.0

1.64 ± 0.12


 para-Nitrophenyl butyrate

3.73 ± 0.11

3.04 ± 0.50

 2-Naphthyl acetate

3.52 ± 0.46

2.53 ± 0.16

Data were obtained with purified proteins

ND no enzymatic activity was detected except spontaneous hydrolysis regardless of the high enzyme loading (2 mg/mL)

Docking studies

In parallel with AbOPHI211A, the alanine residue in position 211 was also found in many other enzymes with a high OPH activity (Luo et al. 2014; Dong et al. 2005). For example, a lactonase was actually engineered into an efficient OPH (PoOPH) without mutating the alanine residue to other residues (Luo et al. 2014). To investigate the possible mechanism of the improved OPH activity caused by I211A mutation, the MP molecule was docked into AbOPHI211A and AbOPH, respectively, to simulate the protein–ligand interaction. In the AbOPHI211A–MP complex (Fig. 3a), the phosphoryl sulfur atom of the substrate MP directly faces the catalytic β zinc atom with a distance of 2.7 Å. The p-nitrophenyl moiety forms π–π interactions with both benzene ring of Phe127 and the indole group of Tyr283, stabilizing the p-nitrophenyl in the leaving group pocket. The nitro group of MP is hydrogen bonded to Asn71 (2.6 Å). One of the O-methyl substituents binds to the His157, implying a strong hydrogen bond between the O-methyl substituent and His157 (2.5 Å). The conformation got from docking simulation implies that the substrate can form strong binding interactions with the catalytic zinc ion and the residues in the binding pocket, which is consistent with the previously catalytic assumptions (Luo et al. 2014; Gotthard et al. 2013).
Fig. 3

Docking pose of MP in the structure of AbOPH or AbOPHI211A. The substrate MP is shown as blue stick. a Details of the interaction of AbOPHI211A with MP in the active site. b Superposition of AbOPH–MP (yellow) and AbOPHI211A–MP (blue). The green arrow represents the different conformation of side chain of His157 in AbOPH–MP and in the AbOPHI211A–MP complex

Residue Ile211 exhibits a distance of 9.7 Å to the β zinc atom and it is located in helix α5, just behind four additional residues 206–209. Based on structural alignment (Fig. 3b) of wild-type AbOPH and the mutant AbOPHI211A, the carboxyl group of Ile211 in the AbOPHI211A–MP complex structure represents a motion of 1.2 Å compared with the AbOPH–MP complex. The loop10 of AbOPHI211A moves closer to helix α5 (0.7 Å), and the side chain of His157 located in loop10 moves toward the same direction (1.4 Å) consequently. On the other hand, His157 of AbOPHI211A displays a distance of 2.5 Å to the methoxyl group of MP, which is shorter than 3.1 Å in the AbOPH–MP complex. Enlightened by the conformational alteration, a reasonable explanation for the increased OPH activity was proposed: I211A substitution decreases the size of the side chain, which offers enough space for the motion of the flexible loop10, and the conformational migration in loop10 makes the side chain of His157 move closer to the methoxyl group of MP, generating a much stronger interaction with MP. Therefore, the mutation I211A adjusted the local structure near the binding pocket and stabilizes the substrate binding with the specificity subsite of OPHs.


An “ancestral” enzyme, AbOPH, was identified with high lactonase activity and faint OPH activity. Its crystal structure was resolved. Ile211 was identified as a key residue for acquiring new OPH activity through semi-rational redesign of AbOPH based on the crystal structure and alignment analysis. The OPH activity of single-mutated AbOPHI211A was remarkably enhanced, with a compromise of its native lactonase activity. This result presents the emergence of new enzyme function by a simple mutation strategy and confirms the high possibility that OPH was evolved from its lactonase ancestor.





organophosphorus hydrolases


a putative OPH from Acinetobacter sp.


OPH isolated from Pseudomonas pseudoalcaligenes


a putative OPH from Pseudomonas oleovorans




methyl-parathion hydrolases




phosphotriesterase-like lactonases


para-nitrophenyl butyrate


Authors’ contributions

JC drafted the manuscript and made substantial contributions to acquisition, analysis and interpretation of data; X-JL and JP designed the study and were responsible for the revision of the manuscript; QC conducted the experiments; J-HX and JZ provided experimental guidance. All authors read and approved the final manuscript.


This work was financially supported by the Ministry of Science and Technology, P.R. China (Nos. 2012AA022206C and 2011CB710800) and The Open Project from the State Key Laboratory of Bioorganic and Natural Product Chemistry, Shanghai 200032, China.

Compliance with ethical guidelines

Competing interests The authors declare that they have no competing interests.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (, which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Authors’ Affiliations

State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing, East China University of Science and Technology
Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences


  1. Afriat L, Roodveldt C, Manco G, Tawfik DS (2006) The latent promiscuity of newly identified microbial lactonases is linked to a recently diverged phosphotriesterase. Biochemistry 45:13677–13686View ArticleGoogle Scholar
  2. Bar-Rogovsky H, Hugenmatter A, Tawfik DS (2013) The evolutionary origins of detoxifying enzymes: the mammalian serum paraoxonases (PONs) relate to bacterial homoserine lactonases. J Biol Chem 288:23914–23927View ArticleGoogle Scholar
  3. Ben-David M, Elias M, Filippi JJ, Dunach E, Silman I, Sussman JL, Tawfik DS (2012) Catalytic versatility and backups in enzyme active sites: the case of serum paraoxonase 1. J Mol Biol 418:181–196View ArticleGoogle Scholar
  4. Cheng TC, DeFrank JJ, Rastogi VK (1999) Alteromonas prolidase for organophosphorus G-agent decontamination. Chem Biol Interact 119:455–462View ArticleGoogle Scholar
  5. Dong YJ, Bartlam M, Sun L, Zhou YF, Zhang ZP, Zhang GG, Rao Z, Zhang XE (2005) Crystal structure of methyl parathion hydrolase from Pseudomonas sp. WBC-3. J Mol Biol 353:655–663View ArticleGoogle Scholar
  6. Du FL, Yu HL, Xu JH, Li CX (2014) Enhanced limonene production by optimizing the expression of limonene biosynthesis and MEP pathway genes in E. coli. Bioresour Bioprocess 1:10View ArticleGoogle Scholar
  7. Dumas DP, Caldwell SR, Wild JR, Raushel FM (1989) Purification and properties of the phosphotriesterase from Pseudomonas diminuta. J Biol Chem 264:19659–19665Google Scholar
  8. Elias M, Dupuy J, Merone L, Mandrich L, Porzio E, Moniot S, Rochu D, Lecomte C, Rossi M, Masson P, Manco G, Chabriere E (2008) Structural basis for natural lactonase and promiscuous phosphotriesterase activities. J Mol Biol 379:1017–1028View ArticleGoogle Scholar
  9. Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60:2126–2132View ArticleGoogle Scholar
  10. Gotthard G, Hiblot J, Gonzalez D, Elias M, Chabriere E (2013) Structural and enzymatic characterization of the phosphotriesterase OPHC2 from pseudomonas pseudoalcaligenes. PLoS ONE 8:e77995View ArticleGoogle Scholar
  11. Hawwa R, Larsen SD, Ratia K, Mesecar AD (2009) Structure-based and random mutagenesis approaches increase the organophosphate-degrading activity of a phosphotriesterase homologue from Deinococcus radiodurans. J Mol Biol 393:36–57View ArticleGoogle Scholar
  12. Hong L, Zhang JJ, Zhang SJ, Zhang XE, Zhou NY (2005) Plasmid-borne catabolism of methyl parathion and p-nitrophenolin Pseudomonas sp. strain WBC-3. Biochem Biophys Res Commun 334:1107–1114View ArticleGoogle Scholar
  13. Khersonsky O, Tawfik DS (2005) Structure-reactivity studies of serum paraoxonase PON1 suggest that its native activity is lactonase. Biochemistry 44:6371–6382View ArticleGoogle Scholar
  14. Luo XJ, Kong XD, Zhao J, Zhou JH, Xu JH (2014) Switching a newly discovered lactonase into an efficient and thermostable phosphotriesterase by simple double mutations His250Ile/Ile263Trp. Biotechnol Bioeng 111:1920–1930View ArticleGoogle Scholar
  15. McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ (2007) Phaser crystallographic software. J Appl Crystallogr 40:658–674View ArticleGoogle Scholar
  16. Merone L, Mandrich L, Porzio E, Rossi M, Muller S, Reiter G, Worek F, Manco G (2010) Improving the promiscuous nerve agent hydrolase activity of a thermostable archaeal lactonase. Bioresour Technol 101:9204–9212View ArticleGoogle Scholar
  17. Murshudov GN, Vagin AA, Dodson EJ (1997) Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D 53:240–255View ArticleGoogle Scholar
  18. Omburo GA, Kuo JM, Mullins LS, Raushel FM (1992) Characterization of the zinc binding site of bacterial phosphotriesterase. J Biol Chem 267:13278–13283Google Scholar
  19. Otwinowski Z, Minor W (1997) Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol 276:307–326View ArticleGoogle Scholar
  20. Raushel FM (2002) Bacterial detoxification of organophosphate nerve agents. Curr Opin Microbiol 5:288–295View ArticleGoogle Scholar
  21. Roodveldt C, Tawfik DS (2005) Shared promiscuous activities and evolutionary features in various members of the amidohydrolase superfamily. Biochemistry 44:12728–12736View ArticleGoogle Scholar
  22. Sapozhnikova Y, Bawardi O, Schlenk D (2004) Pesticides and PCBs insediments and fish from the Salton Sea, California, USA. Chemosphere 55:797–809View ArticleGoogle Scholar
  23. Sapozhnikova Y, Zubcov N, Hungerford S, Roy LA, Boicencob N, Zubcovb E, Schlenka D (2005) Evaluation of pesticides and metals in fish of the Dniester River, Moldova. Chemosphere 60:196–205View ArticleGoogle Scholar
  24. Singh BK (2008) Organophosphorus-degrading bacteria: ecology and industrial applications. Nat Rev Microbiol 7:156–164View ArticleGoogle Scholar
  25. Singh BK, Walker A (2006) Microbial degradation of organophosphorus compounds. FEMS Microbiol Rev 30:428–471View ArticleGoogle Scholar
  26. Vyas NK, Nickitenko A, Rastogi VK, Shah SS, Quiocho FA (2010) Structural insights into the dual activities of the nerve agent degrading organophosphate anhydrolase/prolidase. Biochemistry 49:547–559View ArticleGoogle Scholar
  27. Zhang Y, An J, Ye W, Yang G, Qian ZG, Chen HF, Cui L, Feng Y (2012) Enhancing the promiscuous phosphotriesterase activity of a thermostable lactonase (GkaP) for the efficient degradation of organophosphate pesticides. Appl Environ Microbiol 78:6647–6655View ArticleGoogle Scholar


© Chen et al. 2015