title: Structural and functional characterization of deep-sea thermophilic bacteriophage GVE2 HNH endonuclease.
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.
authors.affiliation: Marine Science &Technology Institute Department of Environmental Science and Engineering, Yangzhou University, China.|State Key Laboratory of Microbial Technology, Shandong University, China.|State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China.
authors.forename: Likui
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Institute of Health Sciences and School of Life Science, Anhui University, Hefei, Anhui 230601, China.
authors.forename: Dandan
authors.initials: D
authors.lastname: Xu
authors.affiliation: Marine Science &Technology Institute Department of Environmental Science and Engineering, Yangzhou University, China.
authors.forename: Yanchao
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Marine Science &Technology Institute Department of Environmental Science and Engineering, Yangzhou University, China.
authors.forename: Xinyuan
authors.initials: X
authors.lastname: Zhu
authors.affiliation: Marine Science &Technology Institute Department of Environmental Science and Engineering, Yangzhou University, China.
authors.forename: Mianwen
authors.initials: M
authors.lastname: Rui
authors.affiliation: Marine Science &Technology Institute Department of Environmental Science and Engineering, Yangzhou University, China.
authors.forename: Ting
authors.initials: T
authors.lastname: Wan
authors.affiliation: Beijing Synchrotron Radiation Facility, Institute of High Energy Physics, Chinese Academy of Sciences, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Zheng
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, China.
authors.forename: Yulong
authors.initials: Y
authors.lastname: Shen
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China.
authors.forename: Xiangdong
authors.initials: X
authors.lastname: Chen
authors.affiliation: Department of Biology, University of Waterloo, Canada.
authors.forename: Kesen
authors.initials: K
authors.lastname: Ma
authors.affiliation: Beijing Synchrotron Radiation Facility, Institute of High Energy Physics, Chinese Academy of Sciences, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Gong
mesh_terms: D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000201:enzymology*; D020134:Catalytic Domain; D004720:Endonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008345:Manganese / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D055550:Protein Stability; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016335:Zinc Fingers
chemical_list: D014764:Viral Proteins; D008345:Manganese; D004720:Endonucleases