The xthA gene product of Archaeoglobus fulgidus is an unspecific DNase.
A thermostable enzyme from the hyperthermophilic sulphate-reducing archaeon, Archaeoglobus fulgidus, was expressed and characterized on the assumption that it is homologous to exonuclease III from Escherichia coli. Sequence similarity database searches were performed based on the amino acid sequence of exonuclease III. The 774 bp long gene was isolated from a culture sample and cloned into different vectors. Expression proved successful by transforming pET28_Af_Exo in Origami B(DE3) containing a tRNA plasmid with extra copies of argU, ileY and leuW tRNA genes as a host strain. The lack of thioredoxin reductase (trxB) and glutathione reductase (gor) in Origami B(DE3) allowed formation of disulfide bridges in the cytosol. Purification was performed by heat treatment of the soluble fraction at 80 degrees C for 30 min followed by a two-step ion exchange chromatography. The activity of the enzyme could be maintained. Optimal activity was achieved at 80 degrees C and at a pH of 7. Within the characterization of the protein we could not find any data verifying exonucleolytic activity in the presence of Mg2+ as described [Ankenbauer, W., Laue, F., Sobek, H., & Greif, M. (2000), patent number WO2001023583]. Instead strong DNA binding properties of the enzyme and nicking activities of double stranded DNA comparable to unspecific DNases could be observed. In contrast to exonuclease III from Escherichia coli, the xthA gene product of Archaeoglobus fulgidus is able to degrade supercoiled plasmids and shows no preferences for blunt or recessed 3'-termini of linear double stranded DNA. The enzyme is inhibited by EDTA and shows only weak activity when replacing Mg2+ with Ca2+ ions.
Miertzschke
Mandy
M
Junior research group 'Protein Engineering', Institute of Biochemistry, Faculty of Biology, Pharmacy and Psychology, University of Leipzig/Biotechnological-Biomedical Centre Leipzig, Germany.
Greiner-Stöffele
Thomas
T
D000595:Amino Acid Sequence; D019611:Archaeoglobus fulgidus / Q000235:genetics*; D001483:Base Sequence; D002413:Cations, Divalent / Q000494:pharmacology; D003851:Deoxyribonucleases / Q000737:chemistry / Q000235:genetics*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010957:Plasmids; D011487:Protein Conformation; D019642:RNA, Archaeal / Q000235:genetics; D012343:RNA, Transfer / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D002413:Cations, Divalent; D019642:RNA, Archaeal; D012343:RNA, Transfer; D003851:Deoxyribonucleases