title: Engineering TaqII bifunctional endonuclease DNA recognition fidelity: the effect of a single amino acid substitution within the methyltransferase catalytic site.
abstract: The aim of this study was to improve a useful molecular tool-TaqII restriction endonuclease-methyltransferase-by rational protein engineering, as well as to show an application of our novel method of restriction endonuclease activity modulation through a single amino acid change in the NPPY motif of methyltransferase. An amino acid change was introduced using site-directed mutagenesis into the taqIIRM gene. The mutated gene was expressed in Escherichia coli. The protein variant was purified and characterized. Previously, we described a TspGWI variant with an amino acid change in the methyltransferase motif IV. Here, we investigate a complex, pleiotropic effect of an analogous amino acid change on its homologue-TaqII. The methyltransferase activity is reduced, but not abolished, while TaqII restriction endonuclease can be reactivated by sinefungin, with an increased DNA recognition fidelity. The general method for engineering of the IIS/IIC/IIG restriction endonuclease activity/fidelity is developed along with the generation of an improved TaqII enzyme for biotechnological applications. A successful application of our novel strategy for restriction endonuclease activity/fidelity alteration, based on bioinformatics analyses, mutagenesis and the use of cofactor-analogue activity modulation, is presented.
authors.affiliation: Department of Molecular Biotechnology, Institute for Environmental and Human Health Protection, Division of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308, Gdańsk, Poland. a.zylicz-stachula@ug.edu.pl.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Zylicz-Stachula
authors.affiliation: Department of Molecular Biotechnology, Institute for Environmental and Human Health Protection, Division of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308, Gdańsk, Poland. asia.zebrowska87@gmail.com.
authors.forename: Joanna
authors.initials: J
authors.lastname: Zebrowska
authors.affiliation: Department of Molecular Biotechnology, Institute for Environmental and Human Health Protection, Division of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308, Gdańsk, Poland. edyta_czajkowska@wp.pl.
authors.forename: Edyta
authors.initials: E
authors.lastname: Czajkowska
authors.affiliation: Department of Molecular Biotechnology, Institute for Environmental and Human Health Protection, Division of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308, Gdańsk, Poland. werusia22@wp.pl.
authors.forename: Weronika
authors.initials: W
authors.lastname: Wrese
authors.affiliation: Department of Molecular Biotechnology, Institute for Environmental and Human Health Protection, Division of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308, Gdańsk, Poland. ewa.sulecka@ug.edu.pl.
authors.forename: Ewa
authors.initials: E
authors.lastname: Sulecka
authors.affiliation: Department of Molecular Biotechnology, Institute for Environmental and Human Health Protection, Division of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308, Gdańsk, Poland. piotr.skowron@ug.edu.pl.
authors.forename: Piotr M
authors.initials: PM
authors.lastname: Skowron
mesh_terms: D019943:Amino Acid Substitution; D020134:Catalytic Domain / Q000235:genetics*; D003001:Cloning, Molecular; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D013824:Thermus / Q000201:enzymology
chemical_list: C042367:endodeoxyribonuclease TaqII; D015252:Deoxyribonucleases, Type II Site-Specific
keywords: Gene mutagenesis; Methyltransferase; Restriction endonuclease; Type IIG; Type IIS