title: Thermo-switchable activity of the restriction endonuclease SsoII achieved by site-directed enzyme modification.
abstract: In this work, the possibility of constructing a thermo-switchable enzyme according to the "molecular gate" strategy is demonstrated. The approach is based on the covalent attachment of oligodeoxyribonucleotides to cysteine residues of an enzyme adjacent to its active center to form a temporal barrier for enzyme-substrate complex formation. The activity of the modified enzyme that had been studied here-the restriction endonuclease SsoII (R.SsoII)-was diminished by a factor of 180 at 25 °С that almost abolished the enzymatic activity when compared with the unmodified enzyme. However, heating of the modified enzyme to 45 °С resulted in a 30-fold increase of activity. The activity of unmodified R.SsoII also increased on heating from 25 to 45 °; however, the difference did not exceed a factor of 3-4. The changes in enzymatic activity observed were shown to be reversible for both the unmodified and the modified R.SsoII. Variation of the length and the sequence of the attached oligodeoxyribonucleotides might allow greater modulation of the activity of DNA-protein conjugates.
authors.affiliation: Department of Bioengineering and Bioinformatics, M. V. Lomonosov Moscow State University, Moscow, Russian Federation; Department of Chemistry, and Belozersky Institute of Physico-Chemical Biology, M. V. Lomonosov Moscow State University, Moscow, Russian Federation.
authors.forename: Liudmila A
authors.initials: LA
authors.lastname: Abrosimova
authors.forename: Mayya V
authors.initials: MV
authors.lastname: Monakhova
authors.forename: Anzhela Yu
authors.initials: AY
authors.lastname: Migur
authors.forename: Wende
authors.initials: W
authors.lastname: Wolfgang
authors.forename: Alfred
authors.initials: A
authors.lastname: Pingoud
authors.forename: Elena A
authors.initials: EA
authors.lastname: Kubareva
authors.forename: Tatiana S
authors.initials: TS
authors.lastname: Oretskaya
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D004247:DNA / Q000737:chemistry; D053837:DNA Cleavage; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000235:genetics; D004789:Enzyme Activation; D004795:Enzyme Stability; D006868:Hydrolysis; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D012764:Shigella sonnei / Q000201:enzymology
chemical_list: D001426:Bacterial Proteins; D004247:DNA; C045297:CCNGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific
keywords: DNA-protein conjugates; restriction endonuclease; thermo-switch