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

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.


Purification, conformation change, and thermostability of the wild type and mutant GVE2 HNHEs.(A) SDS-PAGE of the purified GVE2 HNHE and its H93A, N109A and H118A mutants. (B) Secondary structure assays of the wild type and mutant GVE2 HNHEs by CD analysis. Changes in secondary structure were monitored by scanning from 200 to 250 nm and the mean residue ellipticity was recorded with different line patterns for the wild type and mutant proteins as indicated. (C) Thermal stability assay of the wild type and mutant GVE2 HNHEs by CD analysis. The thermal unfolding of the proteins was determined at 222 nm as described under “Methods”. The melting curves of the wild type and mutant proteins are shown in different line patterns as indicated.
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f4: Purification, conformation change, and thermostability of the wild type and mutant GVE2 HNHEs.(A) SDS-PAGE of the purified GVE2 HNHE and its H93A, N109A and H118A mutants. (B) Secondary structure assays of the wild type and mutant GVE2 HNHEs by CD analysis. Changes in secondary structure were monitored by scanning from 200 to 250 nm and the mean residue ellipticity was recorded with different line patterns for the wild type and mutant proteins as indicated. (C) Thermal stability assay of the wild type and mutant GVE2 HNHEs by CD analysis. The thermal unfolding of the proteins was determined at 222 nm as described under “Methods”. The melting curves of the wild type and mutant proteins are shown in different line patterns as indicated.

Mentions: As discussed above, residues H93, N109 and H118 in GVE2 HNHE are key amino acid residues for DNA nicking. 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 work21. The purified wild type and mutant GVE2 HNHEs are shown in Fig. 4A.


Structural and functional characterization of deep-sea thermophilic bacteriophage GVE2 HNH endonuclease
Purification, conformation change, and thermostability of the wild type and mutant GVE2 HNHEs.(A) SDS-PAGE of the purified GVE2 HNHE and its H93A, N109A and H118A mutants. (B) Secondary structure assays of the wild type and mutant GVE2 HNHEs by CD analysis. Changes in secondary structure were monitored by scanning from 200 to 250 nm and the mean residue ellipticity was recorded with different line patterns for the wild type and mutant proteins as indicated. (C) Thermal stability assay of the wild type and mutant GVE2 HNHEs by CD analysis. The thermal unfolding of the proteins was determined at 222 nm as described under “Methods”. The melting curves of the wild type and mutant proteins are shown in different line patterns as indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Purification, conformation change, and thermostability of the wild type and mutant GVE2 HNHEs.(A) SDS-PAGE of the purified GVE2 HNHE and its H93A, N109A and H118A mutants. (B) Secondary structure assays of the wild type and mutant GVE2 HNHEs by CD analysis. Changes in secondary structure were monitored by scanning from 200 to 250 nm and the mean residue ellipticity was recorded with different line patterns for the wild type and mutant proteins as indicated. (C) Thermal stability assay of the wild type and mutant GVE2 HNHEs by CD analysis. The thermal unfolding of the proteins was determined at 222 nm as described under “Methods”. The melting curves of the wild type and mutant proteins are shown in different line patterns as indicated.
Mentions: As discussed above, residues H93, N109 and H118 in GVE2 HNHE are key amino acid residues for DNA nicking. 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 work21. The purified wild type and mutant GVE2 HNHEs are shown in Fig. 4A.

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.