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Identification of the metal-binding sites of restriction endonucleases by Fe-mediated oxidative cleavage

Biochemistry, ISSN: 0006-2960, Vol: 39, Issue: 11, Page: 3097-3105
2000
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Article Description

Fenton chemistry [Fenton (1894) J. Chem. Soc. 65, 899-910] techniques were employed to identify the residues involved in metal binding located at the active sites of restriction endonucleases. This process uses transition metals to catalytically oxidize the peptide linkage that is in close proximity to the amino acid residues involved in metal ligation. Fe was used as the redox-active transition metal. It was expected that Fe would bind to the endonucleases at the Mg-binding site [Liaw et al. (1993) Biochemistry 32, 7999-4003; Ermacora et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6383-6387; Soundar and Colman (1993) J. Biol. Chem. 268, 5264- 5271; Wei et al. (1994) Biochemistry 33, 7931-7936; Ettner et al. (1995) Biochemistry 34, 22-31; Hlavaty and Nowak (1997) Biochemistry 36, 15515- 15525). Fe-mediated oxidation was successfully performed on TaqI endonuclease, suggesting that this approach could be applied to a wide array of endonucleases [Cao and Barany (1998) J. Biol. Chem. 273, 3300233010]. The restriction endonucleases BamHI, FokI, BglI, BglII, PvuII, SfiI, BssSI, BsoBI, EcoRI, EcoRV, MspI, and HinP1I were subjected to oxidizing conditions in the presence of Fe and ascorbate. All proteins were inactivated upon treatment with Fe and ascorbate. BamHI, FokI, BglI, BglII, PvuII, SfiI, BssSI, and BsoBI were specifically cleaved upon treatment with Fe/ascorbate. The site of Fe/ascorbate-induced protein cleavage for each enzyme was determined. The Fe-mediated oxidative cleavage of BamHI occurs between residues Glu77 and Lys78. Glu77 has been shown by structural and mutational studies to be involved in both metal ligation and catalysis [Newman et al. (1995) Science 269, 656-663; Viadiu and Aggarwal (1998) Nat. Struct. Biol. 5, 910-916; Xu and Schildkraut (1991) J. Biol. Chem. 266, 4425- 4429]. The sites of Fe/ascorbate-induced cleavage for PvuII, FokI, BglI, and BsoBI agree with the metal-binding sites identified in their corresponding three-dimensional structures or from mutational studies [Cheng et al. (1994) EMBO J. 13, 3297-3935; Wah et al. (1997) Nature 388, 97-100; Newman et al. (1998) EMBO J. 17, 5466-5476; Ruan et al. (1997) Gene 188, 35- 39]. The metal-binding residues of BglII, SfiI, and BssSI are proposed based on amino acid sequencing of their Fe/ascorbate-generated cleavage fragments. These results suggest that Fenton chemistry may be a useful methodology in identifying amino acids involved in metal binding in endonucleases.

Bibliographic Details

John J. Hlavaty; Jack S. Benner; Linda J. Hornstra; Ira Schildkraut

American Chemical Society (ACS)

Biochemistry, Genetics and Molecular Biology

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