MUTATION AT A SINGLE ACIDIC AMINO-ACID ENHANCES THE HALOPHILIC BEHAVIOR OF MALATE-DEHYDROGENASE FROM HALOARCULA-MARISMORTUI IN PHYSIOLOGICAL SALTS

In a statistical analysis of the amino acid compositions of 26 halophi lic proteins, 24 showed an increase in acidic amino acids and a decrea se in basic ones when compared to their non-halophlic homologues. The role of acidic residues in halophilic adaptation was investigated by s ite-directed mutagenesis of malate dehydrogenase (MalDH) from Haloarcu la marismortui. In all of 40 non-halophilic homologous proteins, the p osition aligned with E243 in halophilic MalDH is occupied by a non-aci dic amino acid, most frequently by arginine. The E243R mutant of halop hilic MalDH was constructed, over-expressed in Escherichia coli, renat ured and purified. Its salt-dependent catalytic activity was not affec ted compared to the wild-type enzyme and both proteins have the same K -m values for their substrates. The resistance to denaturation of the mutant was compared to that of the wild-type protein in different phys iological salt (NaCl or KCl) and temperature conditions and interprete d in terms of classical quasi-thermodynamic parameters. The mutant is more halophilic than the wild-type protein; it is more sensitive to te mperature and requires significantly higher concentrations of NaCl or KCl for equivalent stability. These results highlight the role of acid ic amino acids in halophilic behaviour and are in agreement with a mod el in which these amino acids act cooperatively to organise hydrated i on binding to the protein.