Increasing cleavage specificity and activity of restriction endonuclease KpnI

Vasu K, Nagamalleswari E, Zahran M, Imhof P, Xu SY, Zhu Z, Chan SH, Nagaraja V (2013)

Publication Type: Journal article

Publication year: 2013

Journal

Nucleic Acids Research Oxford University Press

Book Volume: 41

Pages Range: 9812-9824

Journal Issue: 21

DOI: 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 kM 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 Ca 2+-mediated cleavage activity by altering the geometry of the Ca 2+-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. © The Author(s) 2013. Published by Oxford University Press.

Authors with CRIS profile

Petra Imhof

Involved external institutions

Freie Universität Berlin DE Germany (DE) Indian Institute of Science (IISc) IN India (IN) New England Biolabs US United States (USA) (US) New York University (NYU) US United States (USA) (US)

How to cite

APA:

Vasu, K., Nagamalleswari, E., Zahran, M., Imhof, P., Xu, S.-Y., Zhu, Z.,... Nagaraja, V. (2013). Increasing cleavage specificity and activity of restriction endonuclease KpnI. Nucleic Acids Research, 41(21), 9812-9824. https://dx.doi.org/10.1093/nar/gkt734

MLA:

Vasu, Kommireddy, et al. "Increasing cleavage specificity and activity of restriction endonuclease KpnI." Nucleic Acids Research 41.21 (2013): 9812-9824.

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