The amino acidic substitution of cysteine 167 by serine (C167S) in BstVI restriction endonuclease of Bacillus stearothermophilus V affects its conformation and thermostability.
The restriction endonuclease BstVI from Bacillus stearothermophilus V contains three cysteine residues at positions 134, 167 and 180. Titration of Cys residues with DTNB showed that none of them are involved in disulphide bond formation. Cysteine triplets 134 and 167 were modified by recombinant PCR to introduce a serine residue in each case. The mutated genes were cloned into pGEM-T vector and transformed into E. coli JM109. Even though pGEM-T is not designed for expression, the mutant proteins were efficiently expressed in E. coli. The endonuclease carrying the mutation C134S was purified to homogeneity but appeared to be very unstable. In contrast, the C167S mutant enzyme was stable when pure and was studied biochemically. This mutant enzyme was as stable and resistant to protein-denaturing agents as the wild type enzyme. The activity of both enzymes was not affected by preincubations of 2 h at 80 degrees C. A short preincubation at 95 degrees C caused a complete inactivation of the mutant enzyme while the wild type endonuclease retained 30% of its activity. Moreover, the C167S BstVI was more susceptible to be hydrolyzed by proteinase K and trypsine compared to the wild type endonuclease. These results show that the substitution Cys --> Ser at position 167 affects the configuration and thermostability of BstVI restriction endonuclease.
Loyola
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Laboratorio de Microbiología Molecular, Facultad de Química y Biología, Universidad de Santiago de Chile.
Saavedra
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Gómez
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Vásquez
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D019943:Amino Acid Substitution; D001483:Base Sequence; D002413:Cations, Divalent; D003545:Cysteine / Q000737:chemistry*; D017931:DNA Primers; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D010450:Endopeptidases / Q000378:metabolism; D004795:Enzyme Stability; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D016297:Mutagenesis, Site-Directed; D009994:Osmolar Concentration; D011487:Protein Conformation; D011489:Protein Denaturation; D012694:Serine / Q000737:chemistry*; D013438:Sulfhydryl Compounds / Q000378:metabolism
D002413:Cations, Divalent; D017931:DNA Primers; D013438:Sulfhydryl Compounds; D012694:Serine; C034510:CTCGAG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; D010450:Endopeptidases; D003545:Cysteine