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

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ABSTRACT

HNH endonucleases in bacteriophages play a variety of roles in the phage lifecycle as key components of phage DNA packaging machines. The deep-sea thermophilic bacteriophage Geobacillus virus E2 (GVE2) encodes an HNH endonuclease (GVE2 HNHE). Here, the crystal structure of GVE2 HNHE is reported. This is the first structural study of a thermostable HNH endonuclease from a thermophilic bacteriophage. Structural comparison reveals that GVE2 HNHE possesses a typical ββα-metal fold and Zn-finger motif similar to those of HNH endonucleases from other bacteriophages, apart from containing an extra α-helix, suggesting conservation of these enzymes among bacteriophages. Biochemical analysis suggests that the alanine substitutions of the conserved residues (H93, N109 and H118) in the HNH motif of GVE2 HNHE abolished 94%, 60% and 83% of nicking activity, respectively. Compared to the wild type enzyme, the H93A mutant displayed almost the same conformation while the N108A and H118A mutants had different conformations. In addition, the wild type enzyme was more thermostable than the mutants. In the presence of Mn2+ or Zn2+, the wild type enzyme displayed distinct DNA nicking patterns. However, high Mn2+ concentrations were needed for the N109A and H118A mutants to nick DNA while Zn2+ inactivated their nicking activity.

No MeSH data available.


Mn2+- and Zn2+-binding of GVE2 HNHE.The relative changes in the mean residue ellipticity were monitored at 222 nm as described under “Methods”. The hyperbolic curves were plotted by Origin software. The Kd values for Mn2+- and Zn2+-binding of GVE2 HNHE were calculated to be 0.36 ± 0.08 mM and 1.29 ± 0.29 mM, respectively.
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f8: Mn2+- and Zn2+-binding of GVE2 HNHE.The relative changes in the mean residue ellipticity were monitored at 222 nm as described under “Methods”. The hyperbolic curves were plotted by Origin software. The Kd values for Mn2+- and Zn2+-binding of GVE2 HNHE were calculated to be 0.36 ± 0.08 mM and 1.29 ± 0.29 mM, respectively.

Mentions: To reveal the mechanistic process of DNA nicking by GVE2 HNHE in the presence of Mn2+ or Zn2+, we performed the Mn2+- and Zn2+-binding of the enzyme by CD analysis, respectively. Based on the plotting curves, we found that the Kd values for GVE2 HNNE to bind to Mn2+ and Zn2+ are 0.36 ± 0.08 mM and 1.29 ± 0.29 mM (Fig. 8), respectively. This observation suggests that GVE2 HNHE has a higher affinity for binding to Mn2+ than to Zn2+, which is consistent with the above result that Mn2+ is better than Zn2+ for enabling the enzyme to nick DNA.


Structural and functional characterization of deep-sea thermophilic bacteriophage GVE2 HNH endonuclease
Mn2+- and Zn2+-binding of GVE2 HNHE.The relative changes in the mean residue ellipticity were monitored at 222 nm as described under “Methods”. The hyperbolic curves were plotted by Origin software. The Kd values for Mn2+- and Zn2+-binding of GVE2 HNHE were calculated to be 0.36 ± 0.08 mM and 1.29 ± 0.29 mM, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5304195&req=5

f8: Mn2+- and Zn2+-binding of GVE2 HNHE.The relative changes in the mean residue ellipticity were monitored at 222 nm as described under “Methods”. The hyperbolic curves were plotted by Origin software. The Kd values for Mn2+- and Zn2+-binding of GVE2 HNHE were calculated to be 0.36 ± 0.08 mM and 1.29 ± 0.29 mM, respectively.
Mentions: To reveal the mechanistic process of DNA nicking by GVE2 HNHE in the presence of Mn2+ or Zn2+, we performed the Mn2+- and Zn2+-binding of the enzyme by CD analysis, respectively. Based on the plotting curves, we found that the Kd values for GVE2 HNNE to bind to Mn2+ and Zn2+ are 0.36 ± 0.08 mM and 1.29 ± 0.29 mM (Fig. 8), respectively. This observation suggests that GVE2 HNHE has a higher affinity for binding to Mn2+ than to Zn2+, which is consistent with the above result that Mn2+ is better than Zn2+ for enabling the enzyme to nick DNA.

View Article: PubMed Central - PubMed

ABSTRACT

HNH endonucleases in bacteriophages play a variety of roles in the phage lifecycle as key components of phage DNA packaging machines. The deep-sea thermophilic bacteriophage Geobacillus virus E2 (GVE2) encodes an HNH endonuclease (GVE2 HNHE). Here, the crystal structure of GVE2 HNHE is reported. This is the first structural study of a thermostable HNH endonuclease from a thermophilic bacteriophage. Structural comparison reveals that GVE2 HNHE possesses a typical ββα-metal fold and Zn-finger motif similar to those of HNH endonucleases from other bacteriophages, apart from containing an extra α-helix, suggesting conservation of these enzymes among bacteriophages. Biochemical analysis suggests that the alanine substitutions of the conserved residues (H93, N109 and H118) in the HNH motif of GVE2 HNHE abolished 94%, 60% and 83% of nicking activity, respectively. Compared to the wild type enzyme, the H93A mutant displayed almost the same conformation while the N108A and H118A mutants had different conformations. In addition, the wild type enzyme was more thermostable than the mutants. In the presence of Mn2+ or Zn2+, the wild type enzyme displayed distinct DNA nicking patterns. However, high Mn2+ concentrations were needed for the N109A and H118A mutants to nick DNA while Zn2+ inactivated their nicking activity.

No MeSH data available.