Biochemical And Structural Characterization Of The HNH Endonuclease From Deep-sea Thermophilic Bacteriophage GVE2

His-Asn-His?HNH?endonucleases are ubiquitous in bacteria,archaea,eukaryotes,viruses and bacteriophages,possessing an activity of nicking nucleic acid.To date,HNH endonucleases from mesophiles have been widely investigated;however,the biochemical functions and structures of HNH endonucleases from thermophilic bacteriophages have not been reported.The bacteriophage GVE2?Geobacillus virus E2?was isolated from a deep-sea hydrothermal field in the East Pacific.The host of the bacteriophage GVE2 was Geobacillus sp.E263,and its optimal growth temperature was 60?.The genome of GVE2 has been sequenced,encoding an HNH endonuclease.Primarily,we cloned the gene encoding the GVE2 HNH endonuclease into vector pET-3 0a?+?,and then the recombinant protein was purified after induced expression.Secondly,we characterized biochemical properties of the GVE2 HNH endonuclease.We found that recombinant GVE2 HNH endonuclease exhibited non-specific DNA cleavage activity at 60 0C,which is capable of cleaving ? DNA and cccDNA.Further studies showed that the optimal temperature of the GVE2 HNH endonuclease for cleaving DNA was 60?65?.Thermostability experiment revealed that the enzyme retained its DNA cleavage activity even after heated at 100? for 30 min,suggesting the enzyme is a thermostable endonuclease.The GVE2 HNH endonuclease cleaved DNA over a pH spectrum ranging from 5.5 to 9.0,and the optimal pH of 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 Cu²⁺,the GVE2 HNH endonuclease displayed cleavage activity with varied efficiencies with Mn²⁺,Mg²⁺,Ca²⁺,Fe²⁺,Co²⁺,Zn²⁺ and Ni²⁺,and showed the highest enzyme activity with Mn²⁺.In addition,we found that the GVE2 HNH endonuclease activity was inhibited by NaCl.Collaborated with Professor Gong Yong at Institute of High Energy Physics Chinese Academy of Sciences,we solved the crystal structure of GVE2 HNH endonuclease.The results of crystal structre indicated that a conserved ???-metal motif,three conservative residues?H93,N109 and H118?and a zinc-binding site exist in GVE2 HNH endonuclease.Although the crystal structure of GVE2 HNH endonuclease was strikingly similar to that of Gme?Geobacter metallireducens?HNH endonuclease,but difference still occurred in the two enzymes.Furthermore,the crystal structures of GVE2 HNH endonuclease bounded with Mn²⁺ and Zn²⁺were also solved,respectively.The results suggest that the structures of the two complexes are simliar but dinistinct.In order to reveal the catalytic mechanism of the GVE2 HNH endonuclease,we constructed the GVE2 HNH endonuclease H93A,N109A and H118A mutants by using PCR site-directed mutagenesis and purified the mutant proteins CD analysis results showed that the H93A substitution did not change overall structural of GVE2 HNH endonuclease.However,H118A substitution significantly changed secondary structure obviously and N109A substitution caused the moderate change of secondary structure.Compared with the wild type GVE2 HNH endonuclease,the decrease in thermostability in the three mutants suggested that the three residues associated with thermostability closely.Moreover,we determined the cleavage efficiencies of H93A,N109A and H118A mutants,and found that H93A,N109A and H118A substitutions in the GVE2 HNH endonuclease caused 94%,60%and 83%loss of activity of the enzyme,respectively.In addition,we investigated the effect of Mn²⁺ and Zn²⁺ on nicking DNA of both the wild type and mutant GVE2 HNH endonucleases.We found that in the presence of Mn²⁺ and Zn²⁺,the wild type enzyme cleaved DNA,but displayed distinct cleaving patterns.In the presence of Mn²⁺,the H93 A mutant did not cleave DNA while the N109A and H118 A mutants had reduced abilities to nick DNA.By contrast,all the mutants abolished the DNA cleavage ability in the presence of Mn²⁺.Besides,we found that the GVE2 HNH endonuclease had a higher affinity to Mn²⁺ than Zn²⁺,thus providing the basis for why Mn²⁺ is the optimal divalent metal ion for the enzyme to nick DNA.The study revealed the biochemical properties,structural features and catalytic mechanism of a thermostable HNH endonuclease from deep-sea thermophilic bacteriophage GVE2 for the first time,thus serving as a theoretical underpinning for determining the role of the enzyme in lifecycle of the bacteriophage GVE2.In the meantime,our study provided a thermostable HNH endonuclease in molecular biology and biotechnology.