title: Increasing cleavage specificity and activity of restriction endonuclease KpnI.
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 Mg(2+) ion higher than 500 μM mediates promiscuous activity, Ca(2+) 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 Ca(2+)-mediated cleavage but imparting high cleavage fidelity with Mg(2+). 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.
authors: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India, Department of Chemistry, New York University, NY, USA, Department of Physics, Free University Berlin, Arnimallee 14, 14195 Berlin, Germany, New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA and Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560 064, India.
authors: Kommireddy
authors: K
authors: Vasu
authors: Easa
authors: E
authors: Nagamalleswari
authors: Mai
authors: M
authors: Zahran
authors: Petra
authors: P
authors: Imhof
authors: Shuang-Yong
authors: SY
authors: Xu
authors: Zhenyu
authors: Z
authors: Zhu
authors: Siu-Hong
authors: SH
authors: Chan
authors: Valakunja
authors: V
authors: Nagaraja
mesh_terms: D002118:Calcium / Q000737:chemistry; D020134:Catalytic Domain; D053837:DNA Cleavage; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008274:Magnesium / Q000737:chemistry; D056004:Molecular Dynamics Simulation; D009154:Mutation; D013379:Substrate Specificity
chemical_list: D015252:Deoxyribonucleases, Type II Site-Specific; C029312:GGTACC-specific type II deoxyribonucleases; D008274:Magnesium; D002118:Calcium
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