Marked enhancement of Acinetobacter sp. organophosphorus hydrolase activity by a single residue substitution Ile211Ala
© Chen et al. 2015
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.
KeywordsCrystal 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 184.108.40.206) (Cheng et al. 1999; Vyas et al. 2010), phosphotriesterases (PTEs; EC 220.127.116.11) (Omburo et al. 1992), paraoxonases (PONs; EC 18.104.22.168) (Ben-David et al. 2012), phosphotriesterase-like lactonases (PLLs; EC 22.214.171.124) (Afriat et al. 2006), and methyl-parathion hydrolases (MPHs; EC 126.96.36.199) (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 (http://swissmodel.expasy.org/). 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
Crystal structure of AbOPH
Data collection and refinement statistics
Data collection statistics
Rigaku Micromax-007 HF&Raxis IV++
P21 21 21
Unit cell (Å/deg.)
a = 76.8, b = 82.7, c = 99.8, α = β = γ = 90.0
Resolution range (Å)a
Wilson B factor (Å2)
Solvent content (%)
Resolution range (Å)a
R work (%)b
R free (%)b
Bond lengths (Å)
Bond angles (deg.)
Average B factor (Å2)
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
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
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 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
3.73 ± 0.11
3.04 ± 0.50
3.52 ± 0.46
2.53 ± 0.16
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.
a putative OPH from Acinetobacter sp.
OPH isolated from Pseudomonas pseudoalcaligenes
a putative OPH from Pseudomonas oleovorans
- pNPB :
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.
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