ABSTRACT
BACKGROUND: Phosphotriesterases (PTE) are enzymes capable of detoxifying organophosphate-based chemical warfare agents by hydrolysis. One subclass of these enzymes comprises the family of diisopropylfluorophosphatases (DFPases). The DFPase reported here was originally isolated from squid head ganglion of Loligo vulgaris and can be characterized as squid-type DFPase. It is capable of hydrolyzing the organophosphates diisopropylfluorophosphate, soman, sarin, tabun, and cyclosarin. RESULTS: Crystals were grown of both the native and the selenomethionine-labeled enzyme. The X-ray crystal structure of the DFPase from Loligo vulgaris has been solved by MAD phasing and refined to a crystallographic R value of 17.6% at a final resolution of 1.8 A. Using site-directed mutagenesis, we have structurally and functionally characterized essential residues in the active site of the enzyme. CONCLUSIONS: The crystal structure of the DFPase from Loligo vulgaris is the first example of a structural characterization of a squid-type DFPase and the second crystal structure of a PTE determined to date. Therefore, it may serve as a structural model for squid-type DFPases in general. The overall structure of this protein represents a six-fold beta propeller with two calcium ions bound in a central water-filled tunnel. The consensus motif found in the blades of this beta propeller has not yet been observed in other beta propeller structures. Based on the results obtained from mutants of active-site residues, a mechanistic model for the DFP hydrolysis has been developed.
Subject(s)
Esterases/chemistry , Phosphoric Triester Hydrolases , Amino Acid Sequence , Animals , Binding Sites , Crystallography, X-Ray , Decapodiformes , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Conformation , Sequence Homology, Amino AcidABSTRACT
Di-isopropylfluorophosphatase (DFPase) is shown to contain two high-affinity Ca(2+)-binding sites, which are required for catalytic activity and stability. Incubation with chelating agents results in the irreversible inactivation of DFPase. From titrations with Quin 2 [2-([2-[bis(carboxymethyl)amino]-5-methylphenoxy]-methyl)-6-methoxy-8-[bis(carboxymethyl)-amino]quinoline], a lower-affinity site with dissociation constants of 21 and 840 nM in the absence and the presence of 150 mM KCl respectively was calculated. The higher-affinity site was not accessible, indicating a dissociation constant of less than 5.3 nM. Stopped-flow experiments have shown that the dissociation of bound Ca(2+) occurs in two phases, with rates of approx. 1.1 and 0.026 s(-1) corresponding to the dissociation from the low-affinity and high-affinity sites respectively. Dissociation rates depend strongly on temperature but not on ionic strength, indicating that Ca(2+) dissociation is connected with conformational changes. Limited proteolysis, CD spectroscopy, dynamic light scattering and the binding of 8-anilino-1-naphthalenesulphonic acid have been combined to give a detailed picture of the conformational changes induced on the removal of Ca(2+) from DFPase. The Ca(2+) dissociation is shown to result in a primary, at least partly reversible, step characterized by a large decrease in DFPase activity and some changes in enzyme structure and shape. This step is followed by an irreversible denaturation and aggregation of the apo-enzyme. From the temperature dependence of Ca(2+) dissociation and the denaturation results we conclude that the higher-affinity Ca(2+) site is required for stabilizing DFPase's structure, whereas the lower-affinity site is likely to fulfil a catalytic function.
Subject(s)
Calcium/physiology , Esterases/chemistry , Esterases/metabolism , Phosphoric Triester Hydrolases , Animals , Calcium/metabolism , Circular Dichroism , Decapodiformes , Electrophoresis, Polyacrylamide Gel , Kinetics , Protein Binding , Protein Conformation , Spectrometry, Fluorescence , Structure-Activity Relationship , Terbium/metabolismABSTRACT
'Squid-type' diisopropylfluorophosphatases (DFPases), a subclass of the phosphotriesterases, are enzymes capable of hydrolysing organophosphorus nerve agents. To date, no three-dimensional structure of a 'squid-type' DFPase is known. Here, the crystallization of the DFPase originally isolated from head ganglion of the squid Loligo vulgaris is reported. The protein has been heterologously expressed in Escherichia coli, purified to homogeneity and subsequently crystallized. The protein crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 43.1, b = 82.1, c = 86.6 A and one monomer per asymmetric unit. Under cryoconditions (120 K) the crystals diffracted beyond 2.0 A using a Cu rotating-anode X-ray generator.