// archives

Structural biology

This tag is associated with 4 posts

Neutron structure and mechanistic investigations of DFPase

In a new article in the Journal Acta Crystallographica D we discuss comprehensively the neutron diffraction structure of DFPase and studies on the reaction mechanism of this enzyme. Three mechanisms proposed for DFPase will be discussed. This includes the original mechanism proposed in 2001 in which histidine residue 287 activates a water molecule for nucleophilic attack on the substrate’s phosphorus atom which itself is activated by coordination to the catalytic calcium ion. The second mechanism proposed in 2006 and currently view as the correct one, includes a covalent phospho-enzyme intermediate. The third mechanism which is discussed and refuted includes water as the nucleophile directly activated by coordination to the calcium.The article point out that besides important structural investigations (the neutron diffraction structure of DFPase yielded important and indispensable information) also mutagenesis and other mechanistic studies are required to derive enzyme reaction mechanisms.

X-ray structure of perdeuterated DFPase – perdeuteration of enzymes for neutron diffraction

Perdeuteration of proteins is a much discussed strategy to overcome the problems of incoherent scattering caused by hydrogen in neutron diffraction experiments because deuterium displays a significantly lower incoherent scattering cross section compared to the normal hydrogen isotope. Expression of the protein in perdeuterated media is required for this. We report the X-ray structure of perdeuterated DFPase, which displays practically no differences to the hydrogenous structure. B-factors and RMSD values are reported. Even though a very big perdeuterated DFPase crystal was grown, it did not diffract neutrons. Reasons for this unexpected behaviour are discussed. The structure is presented in a new publication in Acta Cryst. F.

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

The catalytic calcium binding-site of the enzymes DFPase displays remarkable similarities to metal binding sites in structurally related proteins like Paraoxonase (PON1), Drug Resistance Protein 35 (Drp35) from S. aureus or the Gluconolactonase XC5397 from Xanthomonas campestris. DFPase mutants targeting calcium binding residues and their structural characterization allow new insights with respect to metal binding and catalytic activity. The results are described in a new publication in Chemico-Biological Interactions (CBI). The article is part of a Special Issue of CBI on the occasion of the 10th International Meeting on Cholinesterases, which took place in Croatia in September 2009.

“Research Highlight” in Nature Chemistry

Our recently published article in JACS on engineering enantioselectivity in DFPase is now a “Research Highlight” in Nature Chemistry. The article is publicly available but a nature account is required.

Reversed Enantioselectivity of DFPase by Rational Design

Based on structural and mechanistic knowledge, mutants of the phosphotriesterase enzmye DFPase were created that reverse the enantioselectivity of the enzyme from the less toxic to the more toxic stereoisomer of G-type nerve agents. The mutants not only feature a reversed enantioselectivity but also increased enzymatic activity compared to the wildtype. The results are described in a new paper published in the Journal of the American Chemical Society (JACS).

The Fish and the Venom

The German newspaper “Frankfurter Rundschau” published an article about DFPase and our neutron structure with the title “Der Fisch und das Gift” (The Fish and the Venom). The article can be found here.

Neutron Structure of the Enzyme DFPase

A new publication in the journal Proceedings of the National Academy of Sciences of the USA (PNAS) describes the neutron diffraction structure of the enzyme DFPase. Neutron diffraction allows the visualization of hydrogen atoms in protein structures and thus the determination of protonation states and orientations of solvent water molecules. The results of the study confirm the proposed reaction mechanism for DFPase and permit the directed introduction of modifications to the enzyme by mutagenesis to enhance both turnover rates and substrate diversity.