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Free keywords:
calcium binding site; DFP hydrolysis; phosphotriesterase; propeller structure; selenomethionine derivative
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 Å. 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 β propeller with two calcium ions bound in a central water-filled tunnel. The consensus motif found in the blades of this β propeller has not yet been observed in other β propeller structures. Based on the results obtained from mutants of active-site residues, a mechanistic model for the DFP hydrolysis has been developed.
