Biochemical characterization of a thermostable HNH endonuclease from deep-sea thermophilic bacteriophage GVE2

Abstract

His-Asn-His (HNH) proteins are a very common family of small nucleic acid-binding proteins that are generally associated with endonuclease activity and are found in all kingdoms of life. Although HNH endonucleases from mesophiles have been widely investigated, the biochemical functions of HNH endonucleases from thermophilic bacteriophages remain unknown. Here, we characterized the biochemical properties of a thermostable HNH endonuclease from deep-sea thermophilic bacteriophage GVE2. The recombinant GVE2 HNH endonuclease exhibited non-specific cleavage activity at high temperature. The optimal temperature of the GVE2 HNH endonuclease for cleaving DNA was 60-65 °C, and the enzyme retained its DNA cleavage activity even after heating at 100 °C for 30 min, suggesting the enzyme is a thermostable endonuclease. The GVE2 HNH endonuclease cleaved DNA over a wide pH spectrum, ranging from 5.5 to 9.0, and the optimal pH for the enzyme activity was 8.0-9.0. Furthermore, the GVE2 HNH endonuclease activity was dependent on a divalent metal ion. While the enzyme is inactive in the presence of Cu(2+), the GVE2 HNH endonuclease displayed cleavage activity of varied efficiency with Mn(2+), Mg(2+), Ca(2+), Fe(2+), Co(2+), Zn(2+), and Ni(2+). The GVE2 HNH endonuclease activity was inhibited by NaCl. This study provides the basis for determining the role of this endonuclease in life cycle of the bacteriophage GVE2 and suggests the potential application of the enzyme in molecular biology and biotechnology.

Structural and functional characterization of deep-sea thermophilic bacteriophage GVE2 HNH endonuclease

“…Residue H118 in GVE2 HNHE lies in the center of the α4-helix at the end of the C-terminal, which is equivalent to the third ‘H’ of the HNH motif. Our previous study revealed that the GVE2 HNHE H93A mutant abolishes DNA nicking activity 21 , supporting the idea that the residue H93 is one of key residues in the active site center of the enzyme.…”

“…Our previous studies suggest that Mn 2+ is the optimal divalent metal ion for GVE2 HNHE to nick DNA 21 . To reveal how the enzyme associates with Mn 2+ , we solved the structure of GVE2 HNHE in the presence of Mn 2+ at 1.53 Å resolution ( Fig.…”

“…To discern the biochemical function of the conserved HNH motif in GVE2 HNHE, we constructed another two mutants of the enzyme: N109A and H118A. Note that the H93A mutant was constructed in our previous work 21 . The purified wild type and mutant GVE2 HNHEs are shown in Fig.…”

“…reported the nicking sites of GVE2 HNHE in the presence of Mg 2+ 4 . Sequence analysis suggests that GVE2 HNHE possesses an active site similar to those in other HNH endonucleases 21 . Our previous studies suggest that the residue H93 in GVE2 HNHE is a critical residue in the active site for nicking DNA 4 .…”

“…An HNH endonuclease from the thermophilic bacteriophage GVE2, a thermophilic and lytic bacteriophage that infects Geobacillus sp. E263 isolated from a deep-sea hydrothermal field in the east Pacific 19 20 , was the first thermostable member of HNH endonucleases from thermophilic bacteriophages to be biochemically characterized 21 . Xu et al .…”