1B96

ANALYSIS OF A MUTATIONAL HOT-SPOT IN THE ECORV RESTRICTION ENDONUCLEASE: A CATALYTIC ROLE FOR A MAIN CHAIN CARBONYL GROUP

Summary for 1B96

• Entry DOI: 10.2210/pdb1b96/pdb
• Descriptor: DNA (5'-D(*AP*AP*AP*GP*AP*TP*AP*TP*CP*TP*T)-3'), RESTRICTION ENDONUCLEASE ECORV (3 entities in total)
• Functional Keywords: endonuclease, restriction, ecorv, hydrolase-dna complex, hydrolase/dna
• Biological source: Escherichia coli
• Total number of polymer chains: 4
• Total formula weight: 63832.76

Authors

Thomas, M.P.,Halford, S.E.,Brady, R.L. (deposition date: 1999-02-19, release date: 1999-02-26, Last modification date: 2023-08-09)

Primary citation

Thomas, M.P.,Brady, R.L.,Halford, S.E.,Sessions, R.B.,Baldwin, G.S.
Structural analysis of a mutational hot-spot in the EcoRV restriction endonuclease: a catalytic role for a main chain carbonyl group.
Nucleic Acids Res., 27:3438-3445, 1999

PubMed 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.

PubMed: 10446231
DOI: 10.1093/nar/27.17.3438

Experimental method

X-RAY DIFFRACTION (2.3 Å)