|1tx3, resolution 2.50Å ()|
|Gene:||HINDIIR, HI0512 (Haemophilus influenzae)|
HINCII BOUND TO COGNATE DNA
The 2.8 A crystal structure of the type II restriction endonuclease HincII bound to Ca(2+) and cognate DNA containing GTCGAC is presented. The DNA is uncleaved, and one calcium ion is bound per active site, in a position previously described as site I in the related blunt cutting type II restriction endonuclease EcoRV [Horton, N. C., Newberry, K. J., and Perona, J. J. (1998) Proc. Natl. Acad. Sci. U.S.A. 95 (23), 13489-13494], as well as that found in other related enzymes. Unlike the site I metal in EcoRV, but similar to that of PvuII, NgoMIV, BamHI, BglII, and BglI, the observed calcium cation is directly ligated to the pro-S(p) oxygen of the scissile phosphate. A calcium ion-ligated water molecule is well positioned to act as the nucleophile in the phosphodiester bond cleavage reaction, and is within hydrogen bonding distance of the conserved active site lysine (Lys 129), as well as the pro-R(p) oxygen of the phosphate group 3' of the scissile phosphate, suggesting possible roles for these groups in the catalytic mechanism. Kinetic data consistent with an important role for the 3'-phosphate group in DNA cleavage by HincII are presented. The previously observed sodium ion [Horton, N. C., Dorner, L. F., and Perona, J. J. (2002) Nat. Struct. Biol. 9, 42-47] persists in the active sites of the Ca(2+)-bound structure; however, kinetic data show little effect on the single-turnover rate of DNA cleavage in the absence of Na(+) ions.
Ca2+ binding in the active site of HincII: implications for the catalytic mechanism., Etzkorn C, Horton NC, Biochemistry. 2004 Oct 26;43(42):13256-70. PMID:15491133
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.
About this Structure
- Etzkorn C, Horton NC. Ca2+ binding in the active site of HincII: implications for the catalytic mechanism. Biochemistry. 2004 Oct 26;43(42):13256-70. PMID:15491133 doi:10.1021/bi0490082
- Horton NC, Dorner LF, Perona JJ. Sequence selectivity and degeneracy of a restriction endonuclease mediated by DNA intercalation. Nat Struct Biol. 2002 Jan;9(1):42-7. PMID:11742344 doi:10.1038/nsb741