title: Rational engineering of a mesohalophilic carbonic anhydrase to an extreme halotolerant biocatalyst.
abstract: Enzymes expressed by highly salt-tolerant organisms show many modifications compared with salt-affected counterparts including biased amino acid and lower α-helix content, lower solvent accessibility and negative surface charge. Here, we show that halotolerance can be generated in an enzyme solely by modifying surface residues. Rational design of carbonic anhydrase II is undertaken in three stages replacing 18 residues in total, crystal structures confirm changes are confined to surface residues. Catalytic activities and thermal unfolding temperatures of the designed enzymes increase at high salt concentrations demonstrating their shift to halotolerance, whereas the opposite response is found in the wild-type enzyme. Molecular dynamics calculations reveal a key role for sodium ions in increasing halotolerant enzyme stability largely through interactions with the highly ordered first Na(+) hydration shell. For the first time, an approach to generate extreme halotolerance, a trait with broad application in industrial biocatalysis, in a wild-type enzyme is demonstrated.
authors.affiliation: Energy Flagship, Commonwealth Scientific and Industrial Research Organisation (CSIRO), GPO Box 1700, Canberra, Australian Capital Territory 2601, Australia.|Land and Water Flagship, Commonwealth Scientific and Industrial Research Organisation (CSIRO), GPO Box 1700, Canberra, Australian Capital Territory 2601, Australia.
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Warden
authors.affiliation: Energy Flagship, Commonwealth Scientific and Industrial Research Organisation (CSIRO), GPO Box 1700, Canberra, Australian Capital Territory 2601, Australia.|Land and Water Flagship, Commonwealth Scientific and Industrial Research Organisation (CSIRO), GPO Box 1700, Canberra, Australian Capital Territory 2601, Australia.
authors.forename: Michelle
authors.initials: M
authors.lastname: Williams
authors.affiliation: Biomedical Manufacturing Program, Commonwealth Scientific and Industrial Research Organisation (CSIRO), 343 Royal Parade, Parkville, Victoria 3052, Australia.
authors.forename: Thomas S
authors.initials: TS
authors.lastname: Peat
authors.affiliation: Biomedical Manufacturing Program, Commonwealth Scientific and Industrial Research Organisation (CSIRO), 343 Royal Parade, Parkville, Victoria 3052, Australia.
authors.forename: Shane A
authors.initials: SA
authors.lastname: Seabrook
authors.affiliation: Biomedical Manufacturing Program, Commonwealth Scientific and Industrial Research Organisation (CSIRO), 343 Royal Parade, Parkville, Victoria 3052, Australia.
authors.forename: Janet
authors.initials: J
authors.lastname: Newman
authors.affiliation: Energy Flagship, Commonwealth Scientific and Industrial Research Organisation (CSIRO), GPO Box 1700, Canberra, Australian Capital Territory 2601, Australia.|Land and Water Flagship, Commonwealth Scientific and Industrial Research Organisation (CSIRO), GPO Box 1700, Canberra, Australian Capital Territory 2601, Australia.
authors.forename: Greg
authors.initials: G
authors.lastname: Dojchinov
authors.affiliation: Energy Flagship, Commonwealth Scientific and Industrial Research Organisation (CSIRO), GPO Box 1700, Canberra, Australian Capital Territory 2601, Australia.|Department of Chemical Engineering, Monash University, Clayton, Victoria 3168, Australia.
authors.forename: Victoria S
authors.initials: VS
authors.lastname: Haritos
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D055162:Biocatalysis; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002417:Cattle; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D006358:Hot Temperature; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D012965:Sodium Chloride / Q000378:metabolism*
chemical_list: D012965:Sodium Chloride; D002256:Carbonic Anhydrases