Metal ions bound at the active site of the junction-resolving enzyme T7 endonuclease I.
T7 endonuclease I is a nuclease that is selective for the structure of the four-way DNA junction. The active site is similar to those of a number of restriction enzymes. We have solved the crystal structure of endonuclease I with a wild-type active site. Diffusion of manganese ions into the crystal revealed two peaks of electron density per active site, defining two metal ion-binding sites. Site 1 is fully occupied, and the manganese ion is coordinated by the carboxylate groups of Asp55 and Glu65, and the main chain carbonyl of Thr66. Site 2 is partially occupied, and the metal ion has a single protein ligand, the remaining carboxylate oxygen atom of Asp55. Isothermal titration calorimetry showed the sequential exothermic binding of two manganese ions in solution, with dissociation constants of 0.58 +/- 0.019 and 14 +/- 1.5 mM. These results are consistent with a two metal ion mechanism for the cleavage reaction, in which the hydrolytic water molecule is contained in the first coordination sphere of the site 1-bound metal ion.
Hadden
Jonathan M
JM
Astbury Centre for Structural Molecular Biology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
Déclais
Anne-Cécile
AC
Phillips
Simon E V
SE
Lilley
David M J
DM
D019943:Amino Acid Substitution; D000596:Amino Acids / Q000737:chemistry; D017123:Bacteriophage T7 / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D002151:Calorimetry; D002412:Cations / Q000378:metabolism; D018360:Crystallography, X-Ray; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008345:Manganese / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011487:Protein Conformation; D012996:Solutions; D013431:Sulfates / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000596:Amino Acids; D002412:Cations; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D008024:Ligands; D012996:Solutions; D013431:Sulfates; D014764:Viral Proteins; D008345:Manganese; D003850:Deoxyribonuclease I