title: Enhancement of DNaseI Salt Tolerance by Mimicking the Domain Structure of DNase from an Extremely Halotolerant Bacterium Thioalkalivibrio sp. K90mix.
abstract: In our previous work we showed that DNaseI-like protein from an extremely halotolerant bacterium Thioalkalivibrio sp. K90mix retained its activity at salt concentrations as high as 4 M NaCl and the key factor allowing this was the C-terminal DNA-binding domain, which comprised two HhH (helix-hairpin-helix) motifs. The further investigations revealed that this domain originated from proteins related to bacterial competence ComEA/ComE proteins. It is likely that in the course of evolution the DNA-binding domain from these proteins was fused to a metallo-β-lactamase superfamily domain. Very likely such domain organization having proteins subsequently "donated" the DNA-binding domain to bacterial DNases. In this study we have mimicked this evolutionary step by fusing bovine DNaseI and DNA-binding domains. We have created two fusions: one harboring the DNA-binding domain of DNaseI-like protein from Thioalkalivibrio sp. K90mix and the second one harboring the DNA-binding domain of bacterial competence protein ComEA from Bacillus subtilis. Both domains enhanced salt tolerance of DNaseI, albeit to different extent. Molecular modeling revealed the essential differences between their interaction with DNA shedding some light on the differences in salt tolerance. In this study we have enhanced salt tolerance of bovine DNaseI; thus, we successfully mimicked the Nature's evolutionary engineering that created the extremely halotolerant bacterial DNase. We have demonstrated that the newly engineered DNaseI variants can be successfully used in applications where activity of the wild type bovine DNaseI is impeded by buffers used.
authors.affiliation: VU Institute of Biotechnology, V.A. Graiciuno 8, LT-02241 Vilnius, Lithuania.|Thermo Fisher Scientific, V.A. Graiciuno 8, LT-02241 Vilnius, Lithuania.
authors.forename: Gediminas
authors.initials: G
authors.lastname: Alzbutas
authors.affiliation: Thermo Fisher Scientific, V.A. Graiciuno 8, LT-02241 Vilnius, Lithuania.
authors.forename: Milda
authors.initials: M
authors.lastname: Kaniusaite
authors.affiliation: Thermo Fisher Scientific, V.A. Graiciuno 8, LT-02241 Vilnius, Lithuania.
authors.forename: Arunas
authors.initials: A
authors.lastname: Lagunavicius
mesh_terms: D000465:Algorithms; D000818:Animals; D001412:Bacillus subtilis / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry; D002417:Cattle; D003001:Cloning, Molecular; D017931:DNA Primers; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D003850:Deoxyribonuclease I / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D020563:Gammaproteobacteria / Q000201:enzymology*; D006801:Humans; D006860:Hydrogen Bonding; D008958:Models, Molecular; D008968:Molecular Conformation; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012492:Salts / Q000737:chemistry*; D012965:Sodium Chloride / Q000737:chemistry; D055672:Static Electricity
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D012492:Salts; D012965:Sodium Chloride; D003850:Deoxyribonuclease I