2voa

Structure of an AP Endonuclease from Archaeoglobus fulgidus

spinqualitylabelsall models 2voa, resolution 1.70Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 2voa is a 4 chain structure with sequence from Archaeoglobus fulgidus and Synthetic construct. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 1.7Å Ligands: OCS Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Function O29675_ARCFU Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMed The major AP endonuclease in Escherichia coli Exonuclease III (ExoIII) is frequently used in gene technology due to its strong exonucleolytic activity. A thermostabilized variant of ExoIII or a homologous enzyme from thermophilic organisms could be most useful for further applications. For this purpose we characterized a nuclease from the hyperthermophilic archaeon Archaeoglobus fulgidus (Af_Exo), which shares 33% overall sequence identity and 55% similarity to ExoIII. The gene coding for this thermostable enzyme was cloned and expressed in E. coli. The purified protein shows a strong Mg(2+)-dependent nicking activity at AP-sites, nicking of undamaged double-stranded (ds) DNA and a weak exonucleolytic activity. A V217G variant of the enzyme was crystallized with decamer ds-DNA molecule, and the three-dimensional structure was determined to 1.7A resolution. Besides our goal to find or produce a thermostable exonuclease, the structural and catalytic data of Af_Exo and a series of mutant proteins, based on the crystal structure, provide new insight into the mechanism of abasic site recognition and repair. Each of the hydrophobic residues Phe 200, Trp 215 and Val 217, forming a binding pocket for the abasic deoxyribose in Af_Exo, were mutated to glycine or serine. By expanding the size of the binding pocket the unspecific endonucleolytic activity is increased. Thus, size and flexibility of the mostly hydrophobic binding pocket have a significant influence on AP-site specificity. We suggest that its tight fitting to the flipped-out deoxyribose allows for a preferred competent binding of abasic sites. In a larger or more flexible pocket however, intact nucleotides more easily bind in a catalytically competent conformation, resulting in loss of specificity. Moreover, with mutations of Phe 200 and Trp 215 we induced a strong exonucleolytic activity on undamaged DNA. Structure and function of the abasic site specificity pocket of an AP endonuclease from Archaeoglobus fulgidus.,Schmiedel R, Kuettner EB, Keim A, Strater N, Greiner-Stoffele T DNA Repair (Amst). 2008 Dec 4. PMID:19015049 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See Also Exonuclease 3D structures

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