The amino acidic substitution of cysteine 167 by serine ((CS)-S-167) in BstVI restriction endonuclease of Bacillus stearothermophilus V affects its conformation and thermostability

The restriction endonuclease BstVI from Bacillus stearothermophilus V conta ins three cysteine residues at positions 134, 167 and 180. Titration of Cys residues with DTNB showed that none of them are involved in disulphide bon d 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 i s not designed for expression, the mutant proteins were efficiently express ed in E. coli. The endonuclease carrying the mutation (CS)-S-134 was purifi ed to homogeneity but appeared to be very unstable. In contrast, the (CS)-S -167 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 prei ncubations of 2 h at 80 degrees C. A short preincubation at 95 degrees C ca used a complete inactivation of the mutant enzyme while the wild type endon uclease retained 30% of its activity. Moreover, the (CS)-S-167 BstVI was mo re susceptible to be hydrolyzed by proteinase K and trypsine compared to th e wild type endonuclease. These results show that the substitution Cys --> Ser at position 167 affects the configuration and thermostability of BstVI restriction endonuclease. (C) Societe francaise de biochimie et biologie mo leculaire / Elsevier, Paris.