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Characterization of the catalytic calcium binding-site in DFPase and comparison with other beta-propeller enzymes

Catalytic calcium binding-site of DFPase

Catalytic calcium binding-site of DFPase

The enzyme DFpase is structurally well characterized. An atomic resolution X-ray structure and a neutron structure are available. In addition to this a number of DFPase mutants were generated and for some of them X-ray structures were determined. What was missing up to now was a systematic investigation of the calcium binding-site of DFPase with respect to catalytic activity and the ability for metal binding. A summary of existing data and three new X-ray structures of mutants changing residues in the calcium binding-site were now published in a paper in the journal Chemico-Biological Interactions. We also deduce rules for activity and metal biding.

A comparison with the structurally related proteins Paraoxonase (PON1), Drug Resistance Protein 35 (Drp35) from S. aureus or the Gluconolactonase XC5397 from Xanthomonas campestris reveal calcium binding sites with highly similar topology but in part different enzymatic activities (PON1 is also a phosphotriesterase but the native substrates of PON1, Drp35 und XC5397 seem to be lactones). The possible applicability of the rules deduced for DFPase to the other proteins is discussed.

Structural characterization of the catalytic calcium binding site in diisopropyl fluorophosphatase (DFPase) and comparison with related β-propeller enzymes.
Blum MM, Chen JC.
Chem. Biol. Interact. 2010; 187(1-3):373-379.

The calcium-dependent phosphotriesterase diisopropyl fluorophosphatase (DFPase) from the squid Loligo vulgaris efficiently hydrolyzes a wide range of organophosphorus nerve agents. The two calcium ions within DFPase play essential roles for its function. The lower affinity calcium ion located at the bottom of the active site participates in the reaction mechanism, while the high affinity calcium in the center of the protein maintains structural integrity of the enzyme. The activity and structures of three DFPase variants targeting the catalytic calcium-binding site are reported (D121E, N120D/N175D/D229N, and E21Q/N120D/N175D/D229N), and the effect of these mutations on the overall structural dynamics of DFPase is examined using molecular dynamics simulations. While D229 is crucial for enzymatic activity, E21 is essential for calcium binding. Although at least two negatively charged side chains are required for calcium binding, the addition of a third charge significantly lowers the activity. Furthermore, the arrangement of these charges in the binding site is important for enzymatic activity. These results, together with earlier mutational, structural, and kinetic studies, show a highly evolved calcium-binding environment, with a specific electrostatic topology crucial for activity. A number of structural homologues of DFPase have been recently identified, including a chimeric variant of Paraoxonase 1 (PON1), drug resistance protein 35 (Drp35) from Staphylococcus aureus and the gluconolactonase XC5397 from Xanthomonas campestris. Surprisingly, despite low sequence identity, these proteins share remarkably similar calcium-binding environments to DFPase.


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