ArticleNucleic acids research2013
Increasing cleavage specificity and activity of restriction endonuclease KpnI.
Article in Nucleic acids research, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 8 papers.
What it found
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Who cites it
8 citing papers in PubMed, 17 citations in OpenAlex.
- Expression and Purification ofFrontiers in microbiology · 2022Article
- Protein Domain Guided Screen for Sequence Specific and Phosphorothioate-Dependent Restriction Endonucleases.Frontiers in microbiology · 2020Article
- Type II Restriction of Bacteriophage DNA With 5hmdU-Derived Base Modifications.Frontiers in microbiology · 2019Article
- Active site residue identity regulates cleavage preference of LAGLIDADG homing endonucleases.Nucleic acids research · 2018Article
- Article
- Expression and purification of a single-chain Type IV restriction enzyme Eco94GmrSD and determination of its substrate preference.Scientific reports · 2015Article
- Type II restriction endonucleases--a historical perspective and more.Nucleic acids research · 2014Review
- Engineering nicking enzymes that preferentially nick 5-methylcytosine-modified DNA.Nucleic acids research · 2014Article
Corrections and comments
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Authors and funding
8 authors at 3 institutions in 3 countries.
Funding
No grant is acknowledged in the PubMed record.
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.
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Registered trials
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