The xthA gene product of Archaeoglobus fulgidus is an unspecific DNase

Abstract

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°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°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 Mg 2+ 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 Mg 2+ with Ca 2+ ions.

Characterization and disruption of exonuclease genes from Streptomyces aureofaciens B96 and S. coelicolor A3(2)

“…A similar attribute was observed for E. coli exonuclease VII (ExoVII), though that one was absolutely specific for ssDNA (Chase and Richardson 1974;Fernández and Sanchez 2002), and in the case of Archaeoglobus fulgidus Af_Exo (Miertzschke and Greiner-Stöffele 2003). DNAase activity of recExoSa was observed in a broad pH range (6.5-10.5) with a maximum at pH 8.0 either in Tris-HCl or phosphate buffer.…”

Crystallization and preliminary X-ray characterization of two thermostable DNA nucleases

“…Cloning and expression of the genes xthA (ExoAf) and Mth0212 (ExoMt) in E. coli as well as protein production and purification of ExoAf and ExoMt was performed as described in Miertzschke & Greiner-Stö ffele (2003) and Pfeifer & Greiner-Stö ffele (2005), respectively. ExoAf and ExoMt were used directly after ion-exchange chromatography without a further concentration step.…”

“…Since cocrystallization was successful, no further DNA molecules were tried. At room temperature or in the absence of Mg 2+ ions, DNA is not degraded by ExoAf (Miertzschke & Greiner-Stö ffele, 2003) or ExoMt (Pfeifer & Greiner-Stö ffele, 2005). These trials resulted in a couple of different crystallization hits for both nucleases.…”

“…As in the case of ExoAf crystals, two ExoMt molecules bind to one 10-mer DNA. ExoMt is also active as a monomer (Miertzschke & Greiner-Stö ffele, 2003). Tetragonal crystals (ExoMt-II) of various shapes were produced by using solvents that were more hydrophobic (Table 1).…”

“…Two homologous nucleases from the thermophilic archea Archaeoglobus fulgidus and Methanothermobacter thermoautrophicus were chosen for this purpose (Miertzschke & Greiner-Stö ffele, 2003;Pfeifer & Greiner-Stö ffele, 2005). The thermostable Mg 2+ -dependent DNA-cleaving enzyme from A. fulgidus (ExoAf) has an activity optimum at 353 K but shows nonspecific DNase activity.…”