Active sites and thermostability of a non-specific nuclease fromYersinia enterocoliticasubsp. palearcticaby site-directed mutagenesis

Abstract

To identify the amino acid residues that are the critical factors contributing to the high catalytic activity and good thermostability of Yersinia enterocolitica subsp. palearctica non-specific nuclease (Y. NSN), in this work, we analyzed the structure and the functional domain of Y. NSN by using bioinformatics software. Disulphide bonds were analyzed by mass spectrometry. Six active site mutants of Y. NSN, including two salt bridge mutants and four disulphide bond mutants, were obtained by site-directed mutagenesis. Their enzyme activity and thermostability were assayed. The results showed that when changing the amino acids of the active site, or breaking the salt bridges and disulphide bonds, the enzyme activity and thermal stability of Y. NSN decreased significantly, compared to those of the wild-type enzyme. It is indicated that the active site, salt bridges and disulphide bonds play an important role in the enzyme activity and thermostability of Y. NSN.

Are the Closely Related Cobetia Strains of Different Species?

“…The DNA/RNA endonuclease belonging to the structural cl00089: NUC superfamily can non-specifically cleave both double- and single-stranded DNA and RNA, whose domain may be present in phosphodiesterases [ 23 ]. Thus, all these enzymes are fundamental for bacteria survival [ 24 , 25 , 26 , 27 , 28 , 29 ]. If the strains Cobetia sp.…”