Role of calcium ions in the structure and function of the di-isopropylfluorophosphatase from Loligo vulgaris.
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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(carbox ymethyl)-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. (+info)
Insights into the reaction mechanism of the diisopropyl fluorophosphatase from Loligo vulgaris by means of kinetic studies, chemical modification and site-directed mutagenesis.
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Kinetic measurements, chemical modification and site-directed mutagenesis have been employed to gain deeper insights into the reaction mechanism of the diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris. Analysis of the kinetics of diisopropyl fluorophosphate hydrolysis reveals optimal enzyme activity at pH >/=8, 35 degrees C and an ionic strength of 500 mM NaCl, where k(cat) reaches a limiting value of 526 s(-1). The pH rate profile shows that full catalytic activity requires the deprotonation of an ionizable group with an apparent pK(a) of 6.82, DeltaH(ion) of 42 kJ/mol and DeltaS(ion) of 9.8 J/mol K at 25 degrees C. Chemical modification of aspartate, glutamate, cysteine, arginine, lysine and tyrosine residues indicates that these amino acids are not critical for catalysis. None of the six histidine residues present in DFPase reacts with diethyl pyrocarbonate (DEPC), suggesting that DEPC has no accessibility to the histidines. Therefore, all six histidine residues have been individually replaced by asparagine in order to identify residues participating in catalysis. Only substitution of H287 renders the enzyme catalytically almost inactive with a residual activity of approx. 4% compared to wild-type DFPase. The other histidine residues do not significantly influence the enzymatic activity, but H181 and H274 seem to have a stabilizing function. These results are indicative of a catalytic mechanism in which H287 acts as a general base catalyst activating a nucleophilic water molecule by the abstraction of a proton. (+info)
Crystal structure of diisopropylfluorophosphatase from Loligo vulgaris.
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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. (+info)
Evolution of an organophosphate-degrading enzyme: a comparison of natural and directed evolution.
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Organophosphate-degrading enzyme from Agrobacterium radiobacter P230 (OPDA) is a recently discovered enzyme that degrades a broad range of organophosphates. It is very similar to OPH first isolated from Pseudomonas diminuta MG. Despite a high level of sequence identity, OPH and OPDA exhibit different substrate specificities. We report here the structure of OPDA and identify regions of the protein that are likely to give it a preference for substrates that have shorter alkyl substituents. Directed evolution was used to evolve a series of OPH mutants that had activities similar to those of OPDA. Mutants were selected for on the basis of their ability to degrade a number of substrates. The mutations tended to cluster in particular regions of the protein and in most cases, these regions were where OPH and OPDA had significant differences in their sequences. (+info)
Trypsin revisited: crystallography AT (SUB) atomic resolution and quantum chemistry revealing details of catalysis.
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A series of crystal structures of trypsin, containing either an autoproteolytic cleaved peptide fragment or a covalently bound inhibitor, were determined at atomic and ultra-high resolution and subjected to ab initio quantum chemical calculations and multipole refinement. Quantum chemical calculations reproduced the observed active site crystal structure with severe deviations from standard stereochemistry and indicated the protonation state of the catalytic residues. Multipole refinement directly revealed the charge distribution in the active site and proved the validity of the ab initio calculations. The combined results confirmed the catalytic function of the active site residues and the two water molecules acting as the nucleophile and the proton donor. The crystal structures represent snapshots from the reaction pathway, close to a tetrahedral intermediate. The de-acylation of trypsin then occurs in true SN2 fashion. (+info)
Growth of Escherichia coli coexpressing phosphotriesterase and glycerophosphodiester phosphodiesterase, using paraoxon as the sole phosphorus source.
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Phosphotriesterases catalyze the hydrolytic detoxification of phosphotriester pesticides and chemical warfare nerve agents with various efficiencies. The directed evolution of phosphotriesterases to enhance the breakdown of poor substrates is desirable for the purposes of bioremediation. A limiting factor in the identification of phosphotriesterase mutants with increased activity is the ability to effectively screen large mutant libraries. To this end, we have investigated the possibility of coupling phosphotriesterase activity to cell growth by using methyl paraoxon as the sole phosphorus source. The catabolism of paraoxon to phosphate would occur via the stepwise enzymatic hydrolysis of paraoxon to dimethyl phosphate, methyl phosphate, and then phosphate. The Escherichia coli strain DH10B expressing the phosphotriesterase from Agrobacterium radiobacter P230 (OpdA) is unable to grow when paraoxon is used as the sole phosphorus source. Enterobacter aerogenes is an organism capable of growing when dimethyl phosphate is the sole phosphorus source. The enzyme responsible for hydrolyzing dimethyl phosphate has been previously characterized as a nonspecific phosphohydrolase. We isolated and characterized the genes encoding the phosphohydrolase operon. The operon was identified from a shotgun clone that enabled E. coli to grow when dimethyl phosphate is the sole phosphorus source. E. coli coexpressing the phosphohydrolase and OpdA grew when paraoxon was the sole phosphorus source. By constructing a short degradative pathway, we have enabled E. coli to use phosphotriesters as a sole source of phosphorus. (+info)
Contribution of the active-site metal cation to the catalytic activity and to the conformational stability of phosphotriesterase: temperature- and pH-dependence.
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Phosphotriesterase (PTE) detoxifies nerve agents and organophosphate pesticides. The two zinc cations of the PTE active centre can be substituted by other transition metal cations without loss of activity. Furthermore, metal-substituted PTEs display differences in catalytic properties. A prerequisite for engineering highly efficient mutants of PTE is to improve their thermostability. Isoelectric focusing, capillary electrophoresis and steady-state kinetics analysis were used to determine the contribution of the active-site cations Zn2+, Co2+ or Cd2+ to both the catalytic activity and the conformational stability of the corresponding PTE isoforms. The three isoforms have different pI values (7.2, 7.5 and 7.1) and showed non-superimposable electrophoretic titration curves. The overall structural alterations, causing changes in functional properties, were found to be related to the nature of the bound cation: ionic radius and ion electronegativity correlate with Km and kcat respectively. In addition, the pH-dependent activity profiles of isoforms were different. The temperature-dependent profiles of activity showed maximum activity at T < or =35 degrees C, followed by an activation phase near 45-48 degrees C and then inactivation which was completed at 60 degrees C. Analysis of thermal denaturation of the PTEs provided evidence that the activation phase resulted from a transient intermediate. Finally, at the optimum activity between pH 8 and 9.4, the thermostability of the different PTEs increased as the pH decreased, and the metal cation modulated stability (Zn2+-, Co2+- and Cd2+-PTE showed different T (m) values of 60.5-67 degrees C, 58-64 degrees C and 53-64 degrees C respectively). Requirements for optimum activity of PTE (displayed by Co2+-PTE) and maximum stability (displayed by Zn2+-PTE) were demonstrated. (+info)
Structure/function analyses of human serum paraoxonase (HuPON1) mutants designed from a DFPase-like homology model.
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Human serum paraoxonase (HuPON1) is a calcium-dependent enzyme that hydrolyzes esters, including organophosphates and lactones, and exhibits anti-atherogenic properties. A few amino acids have been shown to be essential for the enzyme's arylesterase and organophosphatase activities. Until very recently, a three-dimensional model was not available for HuPON1, so functional roles have not been assigned to those residues. Based on sequence-structure alignment studies, we have folded the amino acid sequence of HuPON1 onto the sixfold beta-propeller structure of squid diisopropylfluorophosphatase (DFPase). We tested the validity of this homology model by circular dichroism (CD) spectroscopy and site-directed mutagenesis. Consistent with predictions from the homology model, CD data indicated that the structural composition of purified HuPON1 consists mainly of beta-sheets. Mutants of HuPON1 were assayed for enzymatic activity against phenyl acetate and paraoxon. Substitution of residues predicted to be important for substrate binding (L69, H134, F222, and C284), calcium ion coordination (D54, N168, N224, and D269), and catalytic mechanism of HuPON1 (H285) led to enzyme inactivation. Mutants F222Y and H115W exhibited substrate-binding selectivity towards phenyl acetate and paraoxon, respectively. The homology model presented here is very similar to the recently obtained PON1 crystal structure, and has allowed identification of several residues within the enzyme active site. (+info)