CRYSTAL-STRUCTURES OF ESCHERICHIA-COLI AND SALMONELLA-TYPHIMURIUM 3-ISOPROPYLMALATE DEHYDROGENASE AND COMPARISON WITH THEIR THERMOPHILIC COUNTERPART FROM THERMUS-THERMOPHILUS

The basis of protein stability has been investigated by the structural comparison of themophilic enzymes with their mesophilic counterparts. A number of characteristics have been found that can contribute to th e stabilization of thermophilic proteins, but no one is uniquely capab le of imparting thermostability. The crystal structure of 3-isopropylm alate dehydrogenase (IPMDH) from the mesophiles Escherichia coli and S almonella typhimurium have been determined by the method of molecular replacement using the known structure of the homologous Thermus thermo philus enzyme. The structure of the E. coil enzyme was refined at a re solution of 2.1 Angstrom to an R-factor of 17.3%, that of the S. typhi murium enzyme at 1.7 Angstrom resolution to an R-factor of 19.8%. The three structures were compared to elucidate the basis of the higher th ermostability of the T. thermophilus enzyme. A mutant that created a c avity in the hydrophobic core of the thermophilic enzyme was designed to investigate the importance of packing density for thermostability. The structure of this mutant was analyzed. The main stabilizing featur es in the thermophilic enzyme are an increased number of salt bridges, additional hydrogen bonds, a proportionately larger and more hydropho bic subunit interface, shortened N and C termini and a larger number o f proline residues. The mutation in the hydrophobic core of T. thermop hilus IPMDH resulted in a cavity of 32 Angstrom(3), but no significant effect on the activity and thermostability of the mutant was observed . (C) 1997 Academic Press Limited.