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PDBsum entry 1rvb
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Hydrolase/DNA
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PDB id
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1rvb
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References listed in PDB file
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Key reference
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Title
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Mg2+ binding to the active site of ecorv endonuclease: a crystallographic study of complexes with substrate and product DNA at 2 a resolution.
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Authors
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D.Kostrewa,
F.K.Winkler.
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Ref.
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Biochemistry, 1995,
34,
683-696.
[DOI no: ]
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PubMed id
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Note In the PDB file this reference is
annotated as "TO BE PUBLISHED".
The citation details given above were identified by an automated
search of PubMed on title and author
names, giving a
percentage match of
89%.
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Abstract
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The type II restriction endonuclease EcoRV was crystallized as a complex with
the substrate DNA undecamer AAAGATATCTT (recognition sequence underlined). These
crystals diffract to much better resolution (2 A) than was the case for the
previously reported complex with the decamer GGGATATCCC [Winkler, F. K., Banner,
D. W., Oefner, C., Tsernoglou, D., Brown, R. S., Heathman, S. P., Bryan, R. K.,
Martin, P. D., Petratos, K., & Wilson, K. S. (1993) EMBO J. 12, 1781-1795].
The crystal structure contains one dimer complex in the asymmetric unit and was
solved by molecular replacement. The same kinked DNA conformation characteristic
for enzyme-bound cognate DNA is observed. Crystals, soaked with Mg2+, show the
essential cofactor bound at only one active site of the dimer, and the DNA is
not cleaved. The Mg2+ has one oxygen from the scissile phosphodiester group and
two carboxylate oxygens, one form Asp74 and one from Asp90, in its octahedral
ligand sphere. The scissile phosphodiester group is pulled by 1 A toward the
Mg2+. After substrate cleavage in solution, isomorphous crystals containing the
enzyme--product--Mg2+ complex were obtained. In this structure, each of the
5'-phosphate groups is bound to two Mg2+. The kinked DNA conformation is
essentially maintained, but the two central adenines, 3' to the cleavage sites,
form an unusual cross-strand base stacking. The structures have been refined to
R factors of 0.16 at 2.1-2.0 A resolution maintaining very good stereochemistry.
On the basis of these structures and inspired by recent kinetic data [Vipond, I.
B., & Halford, S. E. (1994) Biochemistry (second paper of three in this
issue)], we have constructed a transition state model with two metals bound to
the scissile phosphorane group.
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Secondary reference #1
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Title
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The crystal structure of ecorv endonuclease and of its complexes with cognate and non-Cognate DNA fragments.
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Authors
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F.K.Winkler,
D.W.Banner,
C.Oefner,
D.Tsernoglou,
R.S.Brown,
S.P.Heathman,
R.K.Bryan,
P.D.Martin,
K.Petratos,
K.S.Wilson.
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Ref.
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Embo J, 1993,
12,
1781-1795.
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PubMed id
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Secondary reference #2
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Title
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Structure and function of restriction endonucleases
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Author
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F.K.Winkler.
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Ref.
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curr opin struct biol, 1992,
2,
93.
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