Mutational and Structural Studies of the Diisopropylfluorophosphatase from Loligo vulgaris Shed New Light on the Catalytic Mechanism of the Enzyme

Abstract

The active site, the substrate binding site, and the metal binding sites of the diisopropylfluorophosphatase (DFPase) from Loligo vulgaris have been modified by means of site-directed mutagenesis to improve our understanding of the reaction mechanism. Enzymatic characterization of mutants located in the major groove of the substrate binding pocket indicates that large hydrophobic side chains at these positions are favorable for substrate turnover. Moreover, the active site residue His287 proved to be beneficial, but not essential, for DFP hydrolysis. In most cases, hydrophobic side chains at position 287 led to significant catalytic activities although reduced relative to the wild-type enzyme. With respect to the Ca-1 binding site, where catalysis occurs, various mutants indicated that the net charge at this calcium-binding site as well as the relative positions of the charged calcium ligands is crucial for catalytic activity. The importance of the electrostatic potential at the active site was furthermore revealed by various mutations of residues lining the interior of the central water-filled tunnel, which traverses the entire protein structure. In this respect, the structural features of residue His181, which is located at the opposite end of the DFPase tunnel relative to the active site, were characterized extensively. It was concluded that a tunnel-spanning hydrogen bond network, which includes a large number of apparently slow exchanging water molecules, relays any modifications in the electrostatics of the system to the active site, thus affecting the catalytic reactivity of the enzyme.

Luciferase a light source for the silica-based optical waveguides (spicules) in the demosponge Suberites domuncula

“…Vol. 66,2009 Research Article BoxII exists, which had been determined to be crucial for the activity of the insect luciferases [25,26,63]. The corresponding BoxI, involving also the AMP-binding pattern [25], has been considered to display a role in the catalysis of the enzyme [64].…”

“…Vol. 66,2009 Research Article mg, respectively, were measured. The emission spectrum of the S. domuncula enzyme revealed a maximum at 548 nm and a minor peak at 590 nm (pH 8.0) (Fig.…”

Magnetic removal of Entamoeba cysts from water using chitosan oligosaccharide-coated iron oxide nanoparticles

“…After using BLAST, the best template for chitinase homology modeling was identified as human chitotriosidase (PDB entry 1GUV_A, resolution =2.35 Å) with 39% homology similarity ( Figure S7), 36 and diisopropyl fluorophosphatase of Loligo vulgaris (PDB entry 2IAX_A, resolution =1.10 Å) was identified as best template for Jacob with 18.2% homology similarity ( Figure S8). 37 Ramachandran plot of chitinase showed 91.1% amino acid residue in a favorable region, 8.0% residue in an additionally allowed region, and 0.9% residue in a disallowed region ( Figure S9A). In contrast, the Ramachandran plot of Jacob showed 71.1% residue in a favorable region, 27.1% residue in an additionally allowed region, and 1.8% residue in a disallowed region ( Figure S9B).…”

Hydrolytic Enzymes for Chemical Warfare Agent Decontamination

“…This mechanism was challenged when mutations of DFPase (H287F and H287L) were generated that still maintained 65 -80% of the wild -type activity [22] . Computational docking of DFP in the DFPase active site also revealed that the orientation of the DFP molecule with the fl uoride -leaving group pointing away from H287 as required for an inline attack of the activated water was energetically unfavourable.…”