Mutation at a single acidic amino acid enhances the halophilic behaviour of malate dehydrogenase from Haloarcula marismortui in physiological salts.

In a statistical analysis of the amino acid compositions of 26 halophilic proteins, 24 showed an increase in acidic amino acids and a decrease in basic ones when compared to their non-halophilic homologues. The role of acidic residues in halophilic adaptation was investigated by site-directed mutagenesis of malate dehydrogenase (MalDH) from Haloarcula marismortui. In all of 40 non-halophilic homologous proteins, the position aligned with E243 in halophilic MalDH is occupied by a non-acidic amino acid, most frequently by arginine. The E243R mutant of halophilic MalDH was constructed, over-expressed in Escherichia coli, renatured and purified. Its salt-dependent catalytic activity was not affected compared to the wild-type enzyme and both proteins have the same Km values for their substrates. The resistance to denaturation of the mutant was compared to that of the wild-type protein in different physiological salt (NaCl or KCl) and temperature conditions and interpreted in terms of classical quasi-thermodynamic parameters. The mutant is more halophilic than the wild-type protein; it is more sensitive to temperature and requires significantly higher concentrations of NaCl or KCl for equivalent stability. These results highlight the role of acidic amino acids in halophilic behaviour and are in agreement with a model in which these amino acids act cooperatively to organise hydrated ion binding to the protein.