1b94 Citations

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

Nucleic Acids Res 27 3438-45 (1999)
Related entries: 1b95, 1b96, 1b97

Cited: 14 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.

Articles - 1b94 mentioned but not cited (6)

  1. DNA conformations and their sequence preferences. Svozil D, Kalina J, Omelka M, Schneider B. Nucleic Acids Res 36 3690-3706 (2008)
  2. Assessment of the optimization of affinity and specificity at protein-DNA interfaces. Ashworth J, Baker D. Nucleic Acids Res 37 e73 (2009)
  3. Small local variations in B-form DNA lead to a large variety of global geometries which can accommodate most DNA-binding protein motifs. Marathe A, Karandur D, Bansal M. BMC Struct Biol 9 24 (2009)
  4. 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)
  5. Protein stability indicates divergent evolution of PD-(D/E)XK type II restriction endonucleases. Fuxreiter M, Simon I. Protein Sci 11 1978-1983 (2002)
  6. 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)


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)

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