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Increasing cleavage specificity and activity of restriction endonuclease KpnI

Vasu, Kommireddy and Nagamalleswari, Easa and Zahran, Mai and Imhof, Petra and Xu, Shuang-yong and Zhu, Zhenyu and Chan, Siu-Hong and Nagaraja, Valakunja (2013) Increasing cleavage specificity and activity of restriction endonuclease KpnI. In: Nucleic Acids Research, 41 (21). pp. 9812-9824.

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Official URL: http://dx.doi.org/10.1093/nar/gkt734

Abstract

Restriction enzyme KpnI is a HNH superfamily endonuclease requiring divalent metal ions for DNA cleavage but not for binding. The active site of KpnI can accommodate metal ions of different atomic radii for DNA cleavage. Although Mg2+ ion higher than 500 mu M mediates promiscuous activity, Ca2+ suppresses the promiscuity and induces high cleavage fidelity. Here, we report that a conservative mutation of the metal-coordinating residue D148 to Glu results in the elimination of the Ca2+-mediated cleavage but imparting high cleavage fidelity with Mg2+. High cleavage fidelity of the mutant D148E is achieved through better discrimination of the target site at the binding and cleavage steps. Biochemical experiments and molecular dynamics simulations suggest that the mutation inhibits Ca2+-mediated cleavage activity by altering the geometry of the Ca2+-bound HNH active site. Although the D148E mutant reduces the specific activity of the enzyme, we identified a suppressor mutation that increases the turnover rate to restore the specific activity of the high fidelity mutant to the wild-type level. Our results show that active site plasticity in coordinating different metal ions is related to KpnI promiscuous activity, and tinkering the metal ion coordination is a plausible way to reduce promiscuous activity of metalloenzymes.

Item Type: Journal Article
Additional Information: Copyright of this article belongs to Oxford University Press.
Department/Centre: Division of Biological Sciences > Microbiology & Cell Biology
Depositing User: Francis Jayakanth
Date Deposited: 26 Dec 2013 05:08
Last Modified: 26 Dec 2013 05:08
URI: http://eprints.iisc.ac.in/id/eprint/48071

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