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Structural analysis of a mutational hot-spot in the EcoRV restriction endonuclease: a catalytic role for a main chain carbonyl group.

Thomas MP, Brady RL, Halford SE, Sessions RB, Baldwin GS
Nucleic Acids Res 27 3438-45 (1999)
Related entries: 1b94, 1b95, 1b97

Cited: 11 times
EuropePMC logo PMID: 10446231

Abstract

Following random mutagenesis of the Eco RV endonuclease, a high proportion of the null mutants carry substitutions at Gln69. Such mutants display reduced rates for the DNA cleavage step in the reaction pathway, yet the crystal structures of wild-type Eco RV fail to explain why Gln69 is crucial for activity. In this study, crystal structures were determined for two mutants of Eco RV, with Leu or Glu at residue 69, bound to specific DNA. The structures of the mutants are similar to the native protein and no function can be ascribed to the side chain of the amino acid at this locus. Instead, the structures of the mutant proteins suggest that the catalytic defect is due to the positioning of the main chain carbonyl group. In the enzyme-substrate complex for Eco RV, the main chain carbonyl of Gln69 makes no interactions with catalytic functions but, in the enzyme-product complex, it coordinates a metal ion bound to the newly liberated 5'-phosphate. This re-positioning may be hindered in the mutant proteins. Molecular dynamics calculations indicate that the metal on the phosphoryl oxygen interacts with the carbonyl group upon forming the pentavalent intermediate during phosphodiester hydrolysis. A main chain carbonyl may thus play a role in catalysis by Eco RV.

Reviews citing this publication (1)

  1. Structure and function of type II restriction endonucleases. Pingoud A, Jeltsch A. Nucleic Acids Res 29 3705-3727 (2001)

Articles citing this publication (10)

  1. A-form conformational motifs in ligand-bound DNA structures. Lu XJ, Shakked Z, Olson WK. J Mol Biol 300 819-840 (2000)
  2. Analyzing protein-DNA recognition mechanisms. Paillard G, Lavery R. Structure 12 113-122 (2004)
  3. The structure of Bacillus subtilis RecU Holliday junction resolvase and its role in substrate selection and sequence-specific cleavage. McGregor N, Ayora S, Sedelnikova S, Carrasco B, Alonso JC, Thaw P, Rafferty J. Structure 13 1341-1351 (2005)
  4. A "moving metal mechanism" for substrate cleavage by the DNA repair endonuclease APE-1. Oezguen N, Schein CH, Peddi SR, Power TD, Izumi T, Braun W. Proteins 68 313-323 (2007)
  5. Assessment of the optimization of affinity and specificity at protein-DNA interfaces. Ashworth J, Baker D. Nucleic Acids Res 37 e73 (2009)
  6. Unusual role of a cysteine residue in substrate binding and activity of human AP-endonuclease 1. Mantha AK, Oezguen N, Bhakat KK, Izumi T, Braun W, Mitra S. J Mol Biol 379 28-37 (2008)
  7. The energetic contribution of induced electrostatic asymmetry to DNA bending by a site-specific protein. Hancock SP, Hiller DA, Perona JJ, Jen-Jacobson L. J Mol Biol 406 285-312 (2011)
  8. Characterization of RNase HII substrate recognition using RNase HII-argonaute chimaeric enzymes from Pyrococcus furiosus. Kitamura S, Fujishima K, Sato A, Tsuchiya D, Tomita M, Kanai A. Biochem J 426 337-344 (2010)
  9. Efficient methodology for the cyclization of linear peptide libraries via intramolecular S-alkylation using Multipin solid phase peptide synthesis. Roberts KD, Lambert JN, Ede NJ, Bray AM. J Pept Sci 12 525-532 (2006)
  10. Metal Ion Binding at the Catalytic Site Induces Widely Distributed Changes in a Sequence Specific Protein-DNA Complex. Sinha K, Sangani SS, Kehr AD, Rule GS, Jen-Jacobson L. Biochemistry 55 6115-6132 (2016)


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