Different reaction mechanisms discussed for DFPase

Knowledge about the reaction mechanism of the enzyme DFPase is a crucial prerequisite for successful directed protein engineering because the mechanism determines the orientation of the substrates in the binding pocket of the enzyme for catalytic turnover. Also residues important for the mechanism can be specifically optimized. For DFPase three different mechanisms were discussed in the past.

When the first X-ray structure of DFPase was published in 2001 (Scharff et al., Structure 9 (2001) 493-502) residue H287 was found to be part of the enzyme’s binding pocket. Mutant H287N only retained minimal residual activity and it was therefore concluded that H287 is activating a water molecule for nucleophilic attack on the substrate’s phosphorus atom. The substrate itself is activated by coordination to the catalytic calcium ion via the phosphoryl oxygen (top scheme in the figure). As mutants like H287F ratain almost full catalytic activity this mechanism was refuted.

It was alternatively proposed that the calcium ion in the catalytic binding site of DFPase activates a directly coordinated water molecule (resulting in a coordinated hydroxide species). This hydroxide ion would then act as the nucleophile to attack the phosphorus atom of the substrate that is also coordinated to the calcium ()middle scheme in the figure). This mechanism was refuted based on the neutron diffraction structure of DFPase that clearly reveals the identity of the coordinated water as a water molecule and not as hydroxide. It is important to mention in this context that the neutron data (and the respective X-ray data for joint refinement) were recorded at room temperature, which is the relevant temperature for catalytic activity. The neutron structure is however compatible to a third mechanism, which was proposed based on isotope labeling, mutational studies and the structure of a protein-inhibitor complex.

Special issue of Acta D

This mechanism (Blum et al., JACS 128 (2006) 12750-12757) identifies the calcium coordinating residue D229 as the active nucleophile. Als an intermediate an instable high-energy phospho-enzyme species is generated, which is subsequently hydrolyzed by water, regenerating the enzyme and releasing the product (bottom scheme in the figure).

The results of the neutron diffraction experiments with DFPase as well as the results of mutational and kinetic studies were now related to each other in the journal Acta Cryst. D. The article is part of a special issue with the title “Neutrons in Biology“. Even though all articles of the issue are worth reading one article is especially recommended: Benno P. Schoenborn, who published the first neutron diffraction structure of a protein (myoglobin) at the end of the 1960s, offers a fascinating overview over more than fourty years of history of the use of neutron in biomolecular research in his article “A history of neutrons in biology: the development of neutron protein crystallography at BNL and LANL“.

Neutron structure and mechanistic studies of diisopropyl fluorophosphatase (DFPase).
Blum MM, Tomanicek S, John H, Hanson L, Rüterjans H, Schoenborn BP, Langan P, Chen JC.
Acta Crystallogr D Biol Crystallogr. 2010; 66(11):1131-1138.
http://dx.doi.org/10.1107/S0907444910034013

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Abstract:
Diisopropyl fluorophosphatase (DFPase) is a calcium-dependent phosphotriesterase that acts on a variety of highly toxic organophosphorus compounds that act as inhibitors of acetylcholinesterase. The mechanism of DFPase has been probed using a variety of methods, including isotopic labelling, which demonstrated the presence of a phosphoenzyme intermediate in the reaction mechanism. In order to further elucidate the mechanism of DFPase and to ascertain the protonation states of the residues and solvent molecules in the active site, the neutron structure of DFPase was solved at 2.2 Å resolution. The proposed nucleophile Asp229 is deprotonated, while the active-site solvent molecule W33 was identified as water and not hydroxide. These data support a mechanism involving direct nucleophilic attack by Asp229 on the substrate and rule out a mechanism involving metal-assisted water activation. These data also allowed for the re-engineering of DFPase through rational design to bind and productively orient the more toxic S stereoisomers of the nerve agents sarin and cyclosarin, creating a modified enzyme with enhanced overall activity and significantly increased detoxification properties.

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.
http://dx.doi.org/10.1016/j.cbi.2010.02.043

Abstract:
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.