The authors have declared that no competing interests exist.
Conceived and designed the experiments: GG JH ME. Performed the experiments: GG JH DG. Analyzed the data: GG JH DG ME EC. Wrote the paper: GG JH ME.
Organophosphates (OPs) are neurotoxic compounds for which current methods of elimination are unsatisfactory; thus bio-remediation is considered as a promising alternative. Here we provide the structural and enzymatic characterization of the recently identified enzyme isolated from
The X-ray structure of OPHC2 has been solved at 2.1 Å resolution. The enzyme is roughly globular exhibiting a αβ/βα topology typical of the metallo-β-lactamase superfamily. Several structural determinants, such as an extended dimerization surface and an intramolecular disulfide bridge, common features in thermostable enzymes, are consistent with its high Tm (97.8°C). Additionally, we provide the enzymatic characterization of OPHC2 against a wide range of OPs, esters and lactones.
OPHC2 possesses a broad substrate activity spectrum, since it hydrolyzes various phosphotriesters, esters, and a lactone. Because of its organophosphorus hydrolase activity, and given its intrinsic thermostability, OPHC2 is an interesting candidate for the development of an OPs bio-decontaminant. Its X-ray structure shed light on its active site, and provides key information for the understanding of the substrate binding mode and catalysis.
Organophosphates (OPs;
Chemical structures of (
OPs pesticides have been massively used since the 1950’s, leading to the fast emergence of microorganisms that are capable of degrading OPs, and that can probably utilize them as carbon and phosphorus source
A recently identified OPH, named OPHC2 (GenBank ID: AJ605330), has been isolated from
OPHC2 has been previously shown to exhibit methyl-parathion hydrolyzing activity
The alignments were performed using the
The protein production and purification was performed as previously explained
Oligmerization state determination was performed using a size exclusion column S75 10/300 GL (GE-Healthcare) calibrated with the Gel Filtration Low Molecular Weight calibration kit (GE-Healthcare) in
Catalytic parameters were evaluated at 25°C, and recorded with a microplate reader (Synergy HT, BioTek, USA) and the Gen5.1 software in a 6.2 mm path length cell for 200 µL reaction in 96-well plate as previously explained
Standard assays were performed in
Circular Dichroïsm (CD) spectra were recorded as previously described
Crystallization was performed as previously published
Crystals were transferred into a cryo-protectant solution composed of the reservoir solution and 20% (v/v) glycerol prior to flash-cooling in liquid nitrogen. X-ray diffraction dataset was collected at 100 K using synchrotron radiation at the Proxima-1 beam line (SOLEIL, Gif-sur-Yvette, France) and a PILATUS-6M detector (DECTRIS, Switzerland). X-ray diffraction data were integrated and scaled with the
Data collection | |
PDB ID | 4LE6 |
Beamline | PROXIMA-1 |
Wavelength (Å) | 0.980 |
Detector | PILATUS 6M |
Oscillation (°) | 0.15 |
Number of frames | 1200 |
Resolution (Å) (last bin) | 2.1 (2.2–2.1) |
Space group | C2 |
Unit-cell parameters (Å) | a = 109.9, b = 63.8,c = 221.3, β = 101.8 |
No. of observed reflections (last bin) | 252270 (24317) |
No. of unique reflections (last bin) | 82530 (9469) |
Completeness (%)(last bin) | 93.7 (82.6) |
Rmeas (%) (last bin) | 6.5 (50.1) |
I/σ(I) (last bin) | 13.67 (3.07) |
Redundancy (last bin) | 3.06 (2.57) |
Mosaicity (°) | 0.508 |
Refinement statistics | |
Rfree/Rwork | 20.99/17.32 |
No. of total model atoms | 19946 |
Ramachandran favored | 93.5 |
Ramachandran outliers | 0.8 |
Generously allowed rotamers | 5.7 |
Rmsd from ideal | |
Bond lengths (Å) | 0.008 |
Bond angles (°) | 1.111 |
The region 168 to 210, lacking from OPHC2 structure, was modelled using
The chemical nature of the bi-metallic center was studied using anomalous X-ray fluorescence. Two datasets were collected consisting of 3600×0.1° at 2.6 and 3.1 Å resolution at respective wavelengths lower (1.2835 Å) and higher (1.2822 Å) than the Zn-K absorption edge. Moreover, the X-ray fluorescence spectrum of OPHC2 crystal has been collected.
Structural comparisons were made using the crystal structures of MPH (PDB ID 1P9E) and AiiA (PDB ID 2A7M). Structure illustrations, analysis and comparisons were performed using
OPHC2 belongs to the metallo-β-lactamase superfamily and shares significant homology with other representatives such as MPH (identity = 48%; similarity = 58.6%), and lower sequence identity with the lactonases AiiA (17–18%) and AiiB (14%) (
Size exclusion chromatography experiments performed on OPHC2 revealed an apparent molecular weight of 58.3 kDa corresponding to an intermediate size between monomeric (32 kDa) and dimeric (64 kDa) forms (
OPHC2 enzyme was initially characterized for its ability to hydrolyze OPs in crude extracts
Substrates | kcat (s−1) | KM (µM) | kcat/KM (M−1s−1) | |
4.05(±0.01)×10−3 | 94±19 | 1.33(±0.92)×101 | ||
3.87(±0.29)×10−1 | 261±56 | 1.48(±0.34)×103 | ||
ND | ND | ND | ||
5.71(±0.33)×10−2 | 21±6 | 2.68(±0.73)×103 | ||
ND | ND | VLH | ||
3.38(±0.19)×10−1 | 114±17 | 2.96(±0.48)×103 | ||
9.03(±1.26)×10−2 | 1620±563 | 5.56(±2.08)×101 | ||
ND | ND | 2.17(±0.08)×101 | ||
3.22(±0.25)×10−2 | 138±48 | 2.33(±0.83)×102 | ||
ND | ND | ND | ||
ND | ND | ND | ||
2.39±0.20 | 403±100 | 5.93(±1.55)×103 |
Roman numbers correspond to the related chemical structure of the substrate presented in
Concomitantly to the phosphotriesterase activity, esterase or lactonase activities are systematically observed in other enzyme superfamilies such as paraoxonases or PLLs
The structure of OPHC2 was solved at 2.1 Å resolution (
A. General representation of the OPHC2 dimer. Monomers are colored in light and dark blue. The two metals of the active site are represented as grey spheres. Enzyme surface is also represented. B. Electrostatic surface of OPHC2 dimer. Positive and negative potentials are colored in blue and red, respectively. The active sites are indicated by black arrows. C. Cartoon representation of an OPHC2 monomer with α helices colored in blue, β sheets in orange and loops in grey. The secondary structures are numbered from the alignment present in Fig. 2B. The bimetallic centre is shown as two grey spheres and the disulphide bridge is shown as red sticks. N- and C-terminal extremities are also indicated. D. Structural comparison of OPHC2 (in blue) with MPH (in salmon) and AiiA (in green). Major differences concern loops sizes and conformations.
The monomer of OPHC2 is roughly globular with overall dimensions of approximately 44 Å×50 Å×37 Å. As for MPH, its structure could be described as an αβ/βα sandwich topology, typical of the metallo-β-lactamase superfamily
The superposition of OPHC2 with MPH and AiiA yields RMSD values for α-carbon atoms of 0.74 Å (over 213 residues) and 2.41 Å (over 105 residues), respectively. While OPHC2 and MPH structures are very similar, major structural differences are visible between OPHC2 and AiiA. These differences mainly concern the loops size and conformations (
The active site of OPHC2 consists of a cavity with two metal cations: one buried (α metal) and one more solvent exposed (β metal). The α metal is coordinated by His294, His144, Asp143 and the Asp247, the latter coordinates also the β metal together with His226, His139, His141 and a water molecule (
The substrate binding pocket is mainly composed of hydrophobic residues. It can be subdivided, based on the MPH structure
OPHC2 enzyme, as observed for MPH
We here show that OPHC2 hydrolyzes a broad range of esters, from phosphotriesters to the lactone dihydrocoumarin. Being isolated as a phosphotriesterase
The similarities between catalytic centers of OPHC2 and others OPHs, however, suggest a similar enzymatic chemistry. The catalytic center is composed of two metals bridging an activated water molecule as observed in MPH and AiiA structures
Additionally, OPHC2 exhibits lactonase activity. Amongst the 12 tested lactones, OPHC2 processes, however, only dihydrocoumarin. Despite the absence of the conserved Tyr residue, characteristic of lactonases in the metallo-β-lactamase superfamily
Finally, we here show that OPHC2 exhibits relatively low catalytic efficiencies against the range of tested substrates (∼103 s−1M−1 against the best substrates). The average catalytic efficiency of enzymes being ∼105 s−1M−1
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We are grateful to Prof. Dan S. Tawfik and Dr. Moshe Goldsmith for the kind gift of CMP -coumarin. We thank the AFMB laboratory (Marseille, France) for the access to protein production and crystallization platforms. We thank also Jeremy Robin and Charlotte Champion for their valuable inputs, and the reviewers for their comments and corrections of our manuscript.