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Information on EC 3.1.8.2 - diisopropyl-fluorophosphatase and Organism(s) Loligo vulgaris and UniProt Accession Q7SIG4
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3.1.8.2 diisopropyl-fluorophosphataseIUBMB Comments
Acts on phosphorus anhydride bonds (such as phosphorus-halide and phosphorus-cyanide) in organophosphorus compounds (including 'nerve gases'). Inhibited by chelating agents; requires divalent cations. Related to EC 3.1.8.1 aryldialkylphosphatase.
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Word Map
- 3.1.8.2
- dfpase
-
organophosphorus
- dfp
- degradation
- squid
- paraoxonase
- sarin
- phosphotriesterases
- paraoxon
- environmental protection
-
loligo
-
opaas
- arylesterase
-
bioscavenger
- vx
- methylphosphonofluoridate
- medicine
- cyclosarin
-
six-bladed
-
carbanilate
- udp-glucosyltransferase
- diisopropylphosphorofluoridate
-
2.8.2.1
- arylsulfotransferase
-
udp-glucuronyltransferase
-
2.3.1.5
- analysis
The taxonomic range for the selected organisms is: Loligo vulgaris
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota
Synonyms
diisopropylfluorophosphatase, somanase, opa anhydrolase, diisopropyl-fluorophosphatase, ptes5, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
DFPase
-
DFPase
di-isopropylphosphorofluoridate fluorohydrolase
-
-
-
-
dialkylfluorophosphatase
-
-
-
-
diisopropyl phosphorofluoridate hydrolase
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-
-
-
diisopropylfluorophosphatase
-
-
-
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diisopropylfluorophosphonate dehalogenase
-
-
-
-
diisopropylphosphofluoridase
-
-
-
-
isopropylphosphorofluoridase
-
-
-
-
OPA anhydrase
-
-
-
-
OPA anhydrolase
-
-
-
-
OPAA
-
-
-
-
OPAA-2
-
-
-
-
organophosphate acid anhydrase
-
-
-
-
organophosphorus acid anhydrolase
-
-
-
-
somanase
-
-
-
-
tabunase
-
-
-
-
DFPase
-
-
-
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
diisopropyl fluorophosphate + H2O = diisopropyl phosphate + fluoride
catalytic mechanism, H287 is required for catalytic activity
-
diisopropyl fluorophosphate + H2O = diisopropyl phosphate + fluoride
calcium coordinating residue D229, in concert with direct substrate activation by the metal ion, renders the phosphorus atom of the substrate susceptible for attack of water, through generation of a phosphoenzyme intermediate
diisopropyl fluorophosphate + H2O = diisopropyl phosphate + fluoride
analysis of the catalytic reaction mechanism of the enzyme. Proposed mechanism for phosphoenzyme intermediate formation involving Asp229 as the nucleophile. (i) Asp229 attacks the phosphorus center of DFP to form a pentavalent intermediate and (ii) the P-F bond dissociates to form a tetrahedral phosphoenzyme intermediate. Hydrolysis of the phosphoenzyme intermediate is not shown. And proposed mechanism for hydrolysis involving an activated water as the nucleophile. (i) Asp229 abstracts a proton from a water molecule either stepwise or in concert as (ii) water attacks the phosphorus center, (iii) Glu21 abstracts a proton either stepwise or in concert as water forms a bond with phosphorus, and (iv) the P-F bond dissociates
diisopropyl fluorophosphate + H2O = diisopropyl phosphate + fluoride
catalytic reaction mechanism involving residue Asp269, molecular dynamics and modelling, overview
diisopropyl fluorophosphate + H2O = diisopropyl phosphate + fluoride
the catalytic mechanism of DFPase is investigated using the hybrid density functional theory method B3LYP with a large quantum chemical model of the active site abstracted from the X-ray crystal structure. For the first step, two different pathways are considered: (1) residue Asp229 as a nucleophile inline attacks on the phosphorus center and (2) an activated water molecule as the nucleophile attacks on the phosphorus center. Both the Asp229 and the activated water molecule are capable of proceeding nucleophilic attack on the substrate in the presence of Ca2+ ion with the associated barriers 14.8 and 6.0 kcal/mol, respectively. The latter is much easier to perform the nucleophile attack. From the phosphoenzyme intermediate with the hexa-coordinated Ca2+, the uncoordinated Glu21 functions as a general base activated an additional water molecule to attack the carbon center of Asp229 and make the phosphate release. Residues Asn120 and Asn175 promote the elimination of the fluoride via donating strong hydrogen bonds. Residue Asp229 plays a dual role during the hydrolysis reaction process, either as a nucleophile or as a general base to activate the water nucleophile. The role of the calcium ion is providing a necessary conformation of the active site, facilitating the nucleophile formation and substrate orientation
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
hydrolysis of phosphoric triester
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
diisopropyl-fluorophosphate fluorohydrolase
Acts on phosphorus anhydride bonds (such as phosphorus-halide and phosphorus-cyanide) in organophosphorus compounds (including 'nerve gases'). Inhibited by chelating agents; requires divalent cations. Related to EC 3.1.8.1 aryldialkylphosphatase.
CAS REGISTRY NUMBER
COMMENTARY
9032-18-2
-
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
(RS)-propan-2-yl methylphosphonofluoridate + H2O
isopropyl phosphate methylphosphonate + fluoride
i.e. sarin
-
-
?
1,2,2-trimethylpropyl methylphosphonofluoridoate + H2O
1,2,2-trimethylpropyl methylphosphonate + fluoride + H+
-
-
-
?
1-methylethyl methylphosphonofluoridoate + H2O
1-methylethyl methylphosphonate + fluoride + H+
-
-
-
?
3-[fluoro(methyl)phosphoryl]oxy-2,2-dimethylbutane + H2O
3,3-dimethylbutan-2-yl methylphosphonate + fluoride
i.e. soman
-
-
?
cyclohexyl methylphosphonofluoridate + H2O
cyclohexyl methylphosphonate + fluoride
i.e. cyclosarin
-
-
?
cyclohexyl methylphosphonofluoridoate + H2O
cyclohexyl methylphosphonate + fluoride + H+
-
-
-
?
cyclohexylmethylphosphonofluoridate + H2O
cyclohexyl methylphosphonate + fluoride
i.e. cyclosarin
-
-
?
diethyl-paraoxon + H2O
diethyl phosphate + 4-nitrophenol
reaction of EC 3.1.8.1, paraoxonase
-
-
?
ethyl dimethylamidocyanophosphate + H2O
ethyl hydrogen dimethylphosphoramidate + HCN
-
-
-
?
diisopropyl fluorophosphate + H2O
diisopropyl phosphate + fluoride
-
-
-
-
?
(S)-sarin + H2O
?
-
the calculated free energy barrier for hydrolysis of (S)-sarin by the mechanism for diiisopropyl fluorophosphate is highly unfavorable. Hydrolysis of (S)-sarin proceeds by a mechanism in which Asp229 could activate an intervening water molecule for nucleophilic attack on the substrate
-
?
diisopropyl fluorophosphate + H2O
diisopropyl phosphate + fluoride
-
-
-
?
diisopropyl fluorophosphate + H2O
diisopropyl phosphate + fluoride
-
-
-
?
diisopropyl fluorophosphate + H2O
diisopropyl phosphate + fluoride
-
The mechanism for hydrolysis of diisopropyl fluorophosphate involves nucleophilic attack by Asp229 on phosphorus to form a pentavalent intermediate. P-F bond dissociation then yields a phosphoacyl enzyme intermediate in the rate-limiting step. A water molecule, coordinated to the catalytic Ca2+, donates a proton to Asp121 and then attacks the tetrahedral phosphoacyl intermediate to liberate the diisopropyl phosphate product
-
?
diisopropyl fluorophosphate + H2O
diisopropyl phosphate + fluoride
room temperature, pH 7.5
-
-
?
additional information
?
-
diisopropyl-fluorophosphatase (DFPase) from Loligo vulgaris is highly stable and robust biocatalyst for the hydrolysis of various chemical warfare agents such as sarin, soman, tabun, but no natural substrate for DFPase has been identified to date
-
-
?
additional information
?
-
the diisopropylfluorophosphatase (DFPase) from the ganglion and brain of Loligo vulgaris acts on P-F bonds present in some organophosphorus pesticides (OPs)
-
-
?
additional information
?
-
-
the diisopropylfluorophosphatase (DFPase) from the ganglion and brain of Loligo vulgaris acts on P-F bonds present in some organophosphorus pesticides (OPs)
-
-
?
additional information
?
-
DFPase from Loligo vulgaris effectively catalyzes the hydrolysis of the bond between phosphorus and the fluoride (or cyanide) leaving group
-
-
?
additional information
?
-
DFPase from Loligo vulgaris effectively catalyzes the hydrolysis of the bond between phosphorus and the fluoride (or cyanide) leaving group
-
-
?
additional information
?
-
the enzyme is not hydrolytically active against compounds with P-O or P-S leaving group bonds, except for some soman derivatives, and shows no efficient hydrolytic activity against lactones or esters
-
-
?
additional information
?
-
the squid phosphotriesterase diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris shows relatively specific substrate preference, efficiently catalyzing the hydrolysis of diisopropyl fluorophosphate (DFP) and G-type nerve agents, including tabun (GA), sarin (GB), soman (GD), and cyclohexyl sarin (GF). The detoxification of the organophosphorous agent is achieved by the hydrolytic reaction producing a phosphate or phosphonate and a fluoride ion. The DFPase from squid central nervous system shows strong preference for the hydrolysis of P-F or P-CN bonds, which are absent in natural compounds
-
-
?
additional information
?
-
-
diisopropyl fluorophosphatase acts on a variety of organophosphorus compounds
-
-
?
additional information
?
-
-
The enzyme also acts as Ca2+-dependent phosphotriesterase. The hydrolytic reaction catalyzed by DFPase leads to the formation of a phosphate or phosphonate and a fluoride ion, resulting in detoxification of the organophosphorus agent. Presence of a phosphoenzyme intermediate in the reaction mechanism, which involves direct nucleophilic attack by Asp229 on the substrate, but no metal-assisted water activation, overview
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
diisopropyl fluorophosphate + H2O
diisopropyl phosphate + fluoride
-
-
-
-
?
diisopropyl fluorophosphate + H2O
diisopropyl phosphate + fluoride
-
-
-
?
diisopropyl fluorophosphate + H2O
diisopropyl phosphate + fluoride
-
-
-
?
additional information
?
-
diisopropyl-fluorophosphatase (DFPase) from Loligo vulgaris is highly stable and robust biocatalyst for the hydrolysis of various chemical warfare agents such as sarin, soman, tabun, but no natural substrate for DFPase has been identified to date
-
-
?
additional information
?
-
the diisopropylfluorophosphatase (DFPase) from the ganglion and brain of Loligo vulgaris acts on P-F bonds present in some organophosphorus pesticides (OPs)
-
-
?
additional information
?
-
-
the diisopropylfluorophosphatase (DFPase) from the ganglion and brain of Loligo vulgaris acts on P-F bonds present in some organophosphorus pesticides (OPs)
-
-
?
additional information
?
-
-
diisopropyl fluorophosphatase acts on a variety of organophosphorus compounds
-
-
?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Ca2+
Ca2+
required for activity, the wild type enzyme contains two Ca2+ ions
Ca2+
required, Ca2+ ion in active site, coordinated to the moiety of DFP, two glutamines (Asn120 and Asn175), two negatively charged residues (Asp229 and Glu21), and two water molecules, thus completes its hepta-coordinated. Therefore, the metal ion plays an essential role in the precise orientation and activation of the substrate. The active site solvent molecule coordinating the catalytic calcium ion is identified as a water molecule other than a hydroxide
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
O,O-dicyclopentylphosphoroamidate
crystallization data, phosphoryl oxygen of inhibitor is directly coordinated to the catalytic calcium ion
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
presence of glucose-mineral-salt (GMS) supplemented with tryptone and 100 mg/l Co2+ ion increases the specific activity of the recombinant enzyme
-
additional information
-
presence of glucose-mineral-salt (GMS) supplemented with tryptone and 100 mg/l Co2+ ion increases the specific activity of the recombinant enzyme
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
2.72
diisopropyl fluorophosphate
wild-type, pH 7.5, 25°C, in nitrogen atmosphere
23.36
diisopropyl fluorophosphate
wild-type, presence of 1 mM O,O-dicyclopentylphosphoroamidate, pH 7.5, 25°C, in nitrogen atmosphere
65.45
diisopropyl fluorophosphate
wild-type, presence of 3 mM O,O-dicyclopentylphosphoroamidate, pH 7.5, 25°C, in nitrogen atmosphere
additional information
additional information
Michaelis-Menten kinetics
-
additional information
additional information
-
effects of pH, temperature and ionic strength on KM are studied
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
208
diisopropyl fluorophosphate
-
pH 8, 35°C, effects of pH, temperature and ionic strength on kcat are studied
2091
diisopropyl fluorophosphate
wild-type, presence of 3 mM O,O-dicyclopentylphosphoroamidate, pH 7.5, 25°C, in nitrogen atmosphere
2107
diisopropyl fluorophosphate
wild-type, pH 7.5, 25°C, in nitrogen atmosphere
2111
diisopropyl fluorophosphate
wild-type, presence of 1 mM O,O-dicyclopentylphosphoroamidate, pH 7.5, 25°C, in nitrogen atmosphere
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.125
O,O-dicyclopentylphosphoroamidate
wild-type, pH 7.5, 25°C, in nitrogen atmosphere
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
110
substrate sarin, pH 8.0, temperature not specified in the publication
143
158
188
198
substrate cyclosarin, pH 8.0, temperature not specified in the publication
225
substrate diisopropyl fluorophosphate, pH 8.0, temperature not specified in the publication
382.39
purified recombinant engineered His-tagged InaV-N-DFPase enzyme, substrate paraoxon, pH 7.2, 30°C
82
substrate soman, pH 8.0, temperature not specified in the publication
additional information
additional information
a maximum number of 53560000 molecules of diisopropyl fluorophosphate is hydrolyzed per minute per whole cell in the presence of glucose-mineral-salt (GMS) supplemented with tryptone and 100 ppm Co2+ ion. 34.69 U/ml with substrate paraoxon, pH 7.2, 30°C
additional information
-
a maximum number of 53560000 molecules of diisopropyl fluorophosphate is hydrolyzed per minute per whole cell in the presence of glucose-mineral-salt (GMS) supplemented with tryptone and 100 ppm Co2+ ion. 34.69 U/ml with substrate paraoxon, pH 7.2, 30°C
additional information
-
pH dependence of the specific activity of the mutant H287N
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
6 - 9
-
the effect of pH 6.0-9.0 on kinetic constants kcat and KM is studied, KM shows no perceivable dependence on pH within this pH range, kcat increases with pH to a limiting value at pH 8.0
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
the phosphotriesterase activity development between PON1, EC 3.1.8.1, and DFPase, EC 3.1.8.2, is investigated by using the hybrid density functional theory method B3LYP. Structure comparisons of evolutionarily related enzymes show that the mutation of Asn270 leads to the catalytic Ca2+ ion indirectly connecting the buried structural Ca2+ ion via hydrogen bonds in DFPase. It can reduce the plasticity of enzymatic structure, and possibly change the substrate preference from paraoxon (preferred substrate of PON1) to DFP (preferred substrate of DFPase), which implies an evolutionary transition from mono- to dinuclear catalytic centers, enzyme catalysis mechanism from an evolutionary perspective, overview
physiological function
physiological function
-
diisopropyl fluorophosphatase is a calcium-dependent phosphotriesterase that acts on a variety of highly toxic organophosphorus compounds, that act as inhibitors of acetylcholinesterase
additional information
physiological function
the enzyme efficiently catalyze the hydrolysis of the substrate diisopropyl fluorophosphate and a wide range of organophosphorus nerve agents, including soman, sarin, and tabun
physiological function
the squid phosphotriesterase diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris shows relatively specific substrate preference, efficiently catalyzing the hydrolysis of diisopropyl fluorophosphate (DFP) and G-type nerve agents, including tabun (GA), sarin (GB), soman (GD), and cyclohexyl sarin (GF). The detoxification of the organophosphorous (OP) agent is achieved by the hydrolytic reaction producing a phosphate or phosphonate and a fluoride ion
additional information
in detoxification of nerve gas compounds or pesticides in the human body, due to non-human origin of the enzyme, immunological reactions occur when it is injected into body. In order to using DFPase as in vivo detoxifying agent, some manipulations to augment of its efficiency and to decrease of immunogenic problems are needed. Modifications such as PEGylation is one of the possible solutions to conquer these problems
additional information
residue Asp229 plays a role in the coordination variation of calcium during the reaction. Quantum mechanical/molecular mechanical umbrella sampling simulations displays that the hydrolysis of diisopropyl fluorophosphate (DFP) and (S)-sarin processes by DFPase presents two different reaction pathways involving nucleophilic attack by Asp229 or an activated water on phosphorus. Modeling of active site and reaction mechanism with nucleophile Asp229 and coordinating Ca2+, detailed overview. Optimized geometries for the intermediates, transition state, and product for the hydrolysis step of DFPase
additional information
residue Asp229 plays a role in the coordination variation of calcium during the reaction. Quantum mechanical/molecular mechanical umbrella sampling simulations displays that the hydrolysis of diisopropyl fluorophosphate (DFP) and (S)-sarin processes by DFPase presents two different reaction pathways involving nucleophilic attack by Asp229 or an activated water on phosphorus. Modeling of active site and reaction mechanism with nucleophile Asp229 and coordinating Ca2+, detailed overview. Optimized geometries for the intermediates, transition state, and product for the hydrolysis step of DFPase
additional information
the phosphotriesterase diisopropyl fluorophosphatase (DFPase) is a calcium-dependent beta-propeller protein. PON1, EC 3.1.8.1, and DFPase, EC 3.1.8.2, seem to employ similar catalytic mechanisms as phosphotriesterase, due to their structural similarities of active sites. The attacking nucleophile for phosphotriester hydrolysis is identified to be an activated water molecule, with the nucleophile attacking the phosphorus center. The E53Q and D269N mutants in PON1 both possess measurable lactonase and paraoxonase activity, and mutation studies combined with related molecular dynamics simulations suggest that the water activated by Glu53 and Asp269 is the most likely attacking nucleophile. Analysis of the rate-determining reaction step of the organophosphorus compound hydrolysis catalyzed both by DFPase and PON1. Structure-function relationship, overview. Active site structure of DFPase (PDB ID 2GVW) and substrate docking
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). DFPA_LOLVU
314
0
35080
Swiss-Prot
other Location (Reliability: 2)
PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
the molecular conformation of DFPase is similar to other phosphotriesterases such as paraoxonase 1 (PON1, EC 3.1.8.1)
?
x * 56847, wild-type enzyme, sequence calculation, x * 55000, recombinant wild-type enzyme, SDS-PAGE, x * 77000, recombinant engineered His-tagged InaV-N-DFPase enzyme, SDS-PAGE
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
side-chain modification
covalent attachment of polyethylene glycol (PEG) chains to enzymes is a prominent solution to overcome these issues. PEGylation enlarges the hydrodynamic radius of proteins and shields its surface. PEG modification increases the stability against proteases, reduces immunogenicity, and delays renal excretion significantly leading to prolonged half-life, reduced side effects, and increased pharmacological efficiency
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
homology modeling and docking of substrates. The two identical hydrophobic isopropyl groups in diisopropyl fluorophosphate bind to two different sub-pockets in the binding-site. Sub-pocket 1 is formed by residues P36, E37, I72, A74 and M90 while sub-pocket 2 is formed by F173, N175, T195, and W244
in complex with inhibitor O,O-dicyclopentylphosphoroamidate. Phosphoryl oxygen of inhibitor is directly coordinated to the catalytic calcium ion
mutant enzymes are crystallized at room temperature by hanging drop vapor diffusion method, using 0.1 M Tris buffer pH 8.5, 2% tacsimate, 16% (w/v) PEG 3350 for mutant E37A/Y144A/R146A/T195M or 0.2 M KCl, 0.05 M HEPES buffer pH 7.5, 35% (w/v) pentaerythriol propoxylate for mutant E37D/Y144A/R16A/T195M
mutant enzymes N120D/N175D/D229N, E21QN120D/N175D/D229N, and D121E are crystallized by hanging drop vapor diffusion method, using 0.1 M MES buffer pH 6.5, 14-20% (w/v) PEG 3350
quantum mechanical/molecular mechanical umbrella sampling simulations. The mechanism for hydrolysis of diisopropyl fluorophosphate involves nucleophilic attack by Asp229 on phosphorus to form a pentavalent intermediate. P-F bond dissociation then yields a phosphoacyl enzyme intermediate in the rate-limiting step. A water molecule, coordinated to the catalytic Ca2+, donates a proton to Asp121 and then attacks the tetrahedral phosphoacyl intermediate to liberate the diisopropyl phosphate product. The calculated free energy barrier for hydrolysis of (S)-sarin by the same mechanism is highly unfavorable. Hydrolysis of (S)-sarin proceeds by a mechanism in which Asp229 could activate an intervening water molecule for nucleophilic attack on the substrate
at 2.2 A resolution, after exchange of available H atoms by using D2O. Collection of time-of-flight wavelength-resolved Laue images
-
by vapour diffusion using a protein solution with 2 mM protein in 10 mM Tris, pH 7.5, 2 mM CaCl2, against a precipitation solution containing 11% PEG 4000 in 0.1 M MES, pH 6.5, at room temperature, X-ray diffraction structure determination and analysis at 2.2 A resolution. Comparison with structures of enzyme mutants, overview
-
hydrodynamic model calculations based on the DFPase crystal structure from the native state enzyme structure in solution by use of different scattering methods, i.e. small-angle neutron scattering, overview
-
structural characterization of a squid-type enzyme, the overall structure of this protein represents a six-fold beta propeller with two calcium ions bound in a central water-filled tunnel
-
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Asp229/Asn120
site-directed mutagenesis, the mutant shows impaired catalytic activity or decreased substrate binding affinity
Asp229/Asn175
site-directed mutagenesis, the mutant shows impaired catalytic activity or decreased substrate binding affinity
D121E
the mutant displays 87% activity compared to the wild type enzyme
D229N/N120D
D229N/N175D
catalytically inactive, no change in the calcium coordinating environment
E21Q/N120D/N175D/D229N
the mutations lead to a loss of calcium binding and enzymatic activity
E37A/Y144A/R146A/T195M
the mutant shows increased turnover number and kcat/Km for diisopropyl fluorophosphate compared to the wild type enzyme
E37D/Y144A/R16A/T195M
the mutant shows increased turnover number and kcat/Km for diisopropyl fluorophosphate compared to the wild type enzyme
Glu21/Asn120
site-directed mutagenesis, the mutant shows impaired catalytic activity or decreased substrate binding affinity
Glu21/Asn175
site-directed mutagenesis, the mutant shows impaired catalytic activity or decreased substrate binding affinity
N120D/N175D/D229N
the mutations lead to a loss of calcium binding and enzymatic activity
H274N
-
slight loss of activity in comparison to wild-type enzyme using pH Stat measurements, no alteration is observed with fluoride assay
additional information
D229N/N120D
catalytically inactive, no change in the calcium coordinating environment
additional information
intracellular production of organophosphorus pesticides (OPs)-degrading enzymes or the use of native bacteria and fungi leads to a low degradation rate of OPs due to a mass transfer issue which reduces the overall catalytic efficiency. To overcome this challenge, DFPase is expressed on the surface of Escherichia coli for the first time by employing the N-terminal domain of the ice nucleation protein (InaV-N) as an anchoring motif. The recombinant DFPase is successfully located on the outer membrane and shows a significant ability for the biodegradation of diisopropylfluorophosphate (DFP) with a specific activity of 500 U/mg of wet cell weight. The recombinant cells can also degrade chlorpyrifos. No enzyme activity is measured by the fluoride ion-selective electrode in the control sample, inner membrane fraction, or cytoplasm fraction of pET-28a-InaV-N-DFPase cells. High potential of the InaV-N anchoring domain to produce an engineered bacterium that can be used in the bioremediation of pesticide-contaminated environments
additional information
-
intracellular production of organophosphorus pesticides (OPs)-degrading enzymes or the use of native bacteria and fungi leads to a low degradation rate of OPs due to a mass transfer issue which reduces the overall catalytic efficiency. To overcome this challenge, DFPase is expressed on the surface of Escherichia coli for the first time by employing the N-terminal domain of the ice nucleation protein (InaV-N) as an anchoring motif. The recombinant DFPase is successfully located on the outer membrane and shows a significant ability for the biodegradation of diisopropylfluorophosphate (DFP) with a specific activity of 500 U/mg of wet cell weight. The recombinant cells can also degrade chlorpyrifos. No enzyme activity is measured by the fluoride ion-selective electrode in the control sample, inner membrane fraction, or cytoplasm fraction of pET-28a-InaV-N-DFPase cells. High potential of the InaV-N anchoring domain to produce an engineered bacterium that can be used in the bioremediation of pesticide-contaminated environments
additional information
the kinetic measurements of enzyme mutants show that the double mutants Glu21/Asn120, Glu21/Asn175, Asp229/Asn120, and Asp229/Asn175 have impaired catalytic activity or decreased substrate binding affinity although their structures provide unchanged calcium coordination environment proving signification of suitable electrostatic effect of the active site for binding and catalysis
additional information
the kinetic measurements of enzyme mutants show that the double mutants Glu21/Asn120, Glu21/Asn175, Asp229/Asn120, and Asp229/Asn175 have impaired catalytic activity or decreased substrate binding affinity although their structures provide unchanged calcium coordination environment proving signification of suitable electrostatic effect of the active site for binding and catalysis
additional information
-
chemical modification of Tyr, Cys, Arg, Lys, Glu and Asp, this residues are not critical for catalysis
additional information
-
preparation of bicontinuous microemulsions made of sugar surfactants as host systems for the DFPase. The microemulsion remains stable in the presence of the enzyme, scattering experiments. DFPase still has high activity in this complex reaction medium, overview
additional information
-
reengineering of DFPase through rational design to bind and productively orient the more toxic SP stereoisomers of the nerve agents sarin and cyclosarin, creating a modified enzyme with enhanced overall activity and significantly increased detoxification properties
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant His-tagged wild-type and engineered enzymes from Escherichia coli strain BL21 (IDE3) pLysS
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant expression of His-tagged wild-type and engineered enzymes in Escherichia coli strain BL21 (IDE3) pLysS
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
environmental protection
medicine
enzyme DFPase can be used as in vivo detoxifying agent for elimination of organophosphorus chemicals, used as pesticides and warfare nerve agent, e.g. sarin, soman, or tabun
environmental protection
enzyme DFPase can be used as in vivo detoxifying agent for elimination of organophosphorus chemicals, used as pesticides and warfare nerve agent, e.g. sarin, soman, or tabun
environmental protection
the engineered bacterium, prepared with an N-terminal domain of the ice nucleation protein (InaV-N) as an anchoring motif on cell surface of expressing bacteria, can be used in the bioremediation of pesticide-contaminated environments
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Scharff, E.I.; Lucke, C.; Fritzsch, G.; Koepke, J.; Hartleib, J.; Dierl, S.; Ruterjans, H.
Crystallization and preliminary X-ray crystallographic analysis of DFPase from Loligo vulgaris
Acta Crystallogr. Sect. D
57
148-149
2001
Loligo vulgaris
Hartleib, J.; Ruterjans, H.
High-yield expression, purification, and characterization of the recombinant diisopropylfluorophosphatase from Loligo vulgaris
Protein Expr. Purif.
21
210-219
2001
Loligo vulgaris
Hartleib, J.; Ruterjans, H.
Insights into the reaction mechanism of the diisopropyl fluorophosphatase from Loligo vulgaris by means of kinetic studies, chemical modification and site-directed mutagenesis
Biochim. Biophys. Acta
1546
312-324
2001
Loligo vulgaris
Katsemi, V.; Lucke, C.; Koepke, J.; Lohr, F.; Maurer, S.; Fritzsch, G.; Ruterjans, H.
Mutational and structural studies of the diisopropylfluorophosphatase from Loligo vulgaris shed new light on the catalytic mechanism of the enzyme
Biochemistry
44
9022-9033
2005
Loligo vulgaris (Q7SIG4), Loligo vulgaris
Blum, M.M.; Koglin, A.; Rueterjans, H.; Schoenborn, B.; Langan, P.; Chen, J.C.
Preliminary time-of-flight neutron diffraction study on diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris
Acta Crystallogr. Sect. F
63
42-45
2007
Loligo vulgaris
Blum, M.M.; Loehr, F.; Richardt, A.; Rueterjans, H.; Chen, J.C.
Binding of a designed substrate analogue to diisopropyl fluorophosphatase: implications for the phosphotriesterase mechanism
J. Am. Chem. Soc.
128
12750-12757
2006
Loligo vulgaris (Q7SIG4)
Gaeb, J.; Melzer, M.; Kehe, K.; Richardt, A.; Blum, M.M.
Quantification of hydrolysis of toxic organophosphates and organophosphonates by diisopropyl fluorophosphatase from Loligo vulgaris by in situ Fourier transform infrared spectroscopy
Anal. Biochem.
385
187-193
2009
Loligo vulgaris
Blum, M.M.; Mustyakimov, M.; Rueterjans, H.; Kehe, K.; Schoenborn, B.P.; Langan, P.; Chen, J.C.
Rapid determination of hydrogen positions and protonation states of diisopropyl fluorophosphatase by joint neutron and X-ray diffraction refinement
Proc. Natl. Acad. Sci. USA
106
713-718
2009
Loligo vulgaris (Q7SIG4)
Blum, M.M.; Tomanicek, S.J.; John, H.; Hanson, B.L.; Rueterjans, H.; Schoenborn, B.P.; Langan, P.; Chen, J.C.
X-ray structure of perdeuterated diisopropyl fluorophosphatase (DFPase): perdeuteration of proteins for neutron diffraction
Acta Crystallogr. Sect. F
66
379-385
2010
Loligo vulgaris (Q7SIG4)
Gaeb, J.; Melzer, M.; Kehe, K.; Wellert, S.; Hellweg, T.; Blum, M.M.
Monitoring the hydrolysis of toxic organophosphonate nerve agents in aqueous buffer and in bicontinuous microemulsions by use of diisopropyl fluorophosphatase (DFPase) with 1H-31P HSQC NMR spectroscopy
Anal. Bioanal. Chem.
396
1213-1221
2010
Loligo vulgaris
Blum, M.M.; Chen, J.C.
Structural characterization of the catalytic calcium-binding site in diisopropyl fluorophosphatase (DFPase)-Comparison with related beta-propeller enzymes
Chem. Biol. Interact.
187
373-379
2010
Loligo vulgaris (Q7SIG4)
Melzer, M.; Chen, J.C.; Heidenreich, A.; Gaeb, J.; Koller, M.; Kehe, K.; Blum, M.M.
Reversed enantioselectivity of diisopropyl fluorophosphatase against organophosphorus nerve agents by rational design
J. Am. Chem. Soc.
131
17226-17232
2009
Loligo vulgaris (Q7SIG4)
Chen, J.C.; Mustyakimov, M.; Schoenborn, B.P.; Langan, P.; Blum, M.M.
Neutron structure and mechanistic studies of diisopropyl fluorophosphatase (DFPase)
Acta Crystallogr. Sect. D
66
1131-1138
2010
Loligo vulgaris
Wellert, S.; Tiersch, B.; Koetz, J.; Richardt, A.; Lapp, A.; Holderer, O.; Gaeb, J.; Blum, M.M.; Schulreich, C.; Stehle, R.; Hellweg, T.
The DFPase from Loligo vulgaris in sugar surfactant-based bicontinuous microemulsions: structure, dynamics, and enzyme activity
Eur. Biophys. J.
40
761-774
2011
Loligo vulgaris
Belinskaya, T.; Pattabiraman, N.; diTargiani, R.; Choi, M.; Saxena, A.
Differences in amino acid residues in the binding pockets dictate substrate specificities of mouse senescence marker protein-30, human paraoxonase1, and squid diisopropylfluorophosphatase
Biochim. Biophys. Acta
1824
701-710
2012
Loligo vulgaris (Q7SIG4)
Wymore, T.; Field, M.J.; Langan, P.; Smith, J.C.; Parks, J.M.
Hydrolysis of DFP and the nerve agent (S)-sarin by DFPase proceeds along two different reaction pathways: Implications for engineering bioscavengers
J. Phys. Chem. B
118
4479-4489
2014
Loligo vulgaris (Q7SIG4)
Latifi, A.M.; Karami, A.; Khodi, S.
Efficient surface display of diisopropylfluorophosphatase (DFPase) in E. coli for biodegradation of toxic organophosphorus compounds (DFP and Cp)
Appl. Biochem. Biotechnol.
177
624-636
2015
Loligo vulgaris (Q7SIG4), Loligo vulgaris
Allahyari, H.; Latifi, A.
Diisopropyl-fluorophosphatase as a catalytic bioscavenger
J. Appl. Biotechnol. Rep.
3
477-482
2016
Loligo vulgaris (Q7SIG4)
-
Zhang, H.; Yang, L.; Ma, Y.Y.; Zhu, C.; Lin, S.; Liao, R.Z.
Theoretical studies on catalysis mechanisms of serum paraoxonase 1 and phosphotriesterase diisopropyl fluorophosphatase suggest the alteration of substrate preference from paraoxonase to DFP
Molecules
23
1660
2018
Loligo vulgaris (Q7SIG4)
Xu, C.; Yang, L.; Yu, J.; Liao, R.
What roles do the residue Asp229 and the coordination variation of calcium play of the reaction mechanism of the diisopropyl-fluorophosphatase? A DFT investigation
Theoret. Chem. Accounts
135
1-11
2016
Loligo vulgaris (Q7SIG4)
-
Xu, C.; Yang, L.; Yu, J.; Liao, R.
What roles do the residue Asp229 and the coordination variation of calcium play of the reaction mechanism of the diisopropyl-fluorophosphatase? A DFT investigation
Theoret. Chem. Accounts
135
138
2016
Loligo vulgaris (Q7SIG4)
-
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