PvuII endonuclease contains two calcium ions in active sites.
Restriction endonucleases differ in their use of metal cofactors despite having remarkably similar folds for their catalytic regions. To explore this, we have characterized the interaction of endonuclease PvuII with the catalytically incompetent cation Ca(2+). The structure of a glutaraldehyde-crosslinked crystal of the endonuclease PvuII-DNA complex, determined in the presence of Ca(2+) at a pH of approximately 6.5, supports a two-metal mechanism of DNA cleavage by PvuII. The first Ca(2+) position matches that found in all structurally examined endonucleases, while the second position is similar to that of EcoRV but is distinct from that of BamHI and BglI. The location of the second metal in PvuII, unlike that in BamHI/BglI, permits no direct interaction between the second metal and the O3' oxygen leaving group. However, the interactions between the DNA scissile phosphate and the metals, the first metal and the attacking water, and the attacking water and DNA are the same in PvuII as they are in the two-metal models of BamHI and BglI, but are distinct from the proposed three-metal or the two-metal models of EcoRV.
Horton
J R
JR
Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road, Atlanta, GA 30322, USA.
Cheng
X
X
D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000378:metabolism; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000378:metabolism*; D005976:Glutaral / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008958:Models, Molecular; D010710:Phosphates / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011514:Proteus vulgaris / Q000201:enzymology*; D013329:Structure-Activity Relationship; D014867:Water / Q000378:metabolism
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D010710:Phosphates; D014867:Water; D008345:Manganese; D004247:DNA; C025630:CAGCTG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; D008274:Magnesium; D002118:Calcium; D005976:Glutaral