Many enzymes exhibit higher catalytic activity and increased thermal stability at moderately high pressures. Enhanced stability can be particularly dramatic for hyperthermophilic enzymes at very high temperatures, i.e., near or above 100°C. Improved stability under pressure thus appears to be related to enzyme thermostability. Studying the properties of proteins adapted to both high pressure and temperature should illuminate favorable interactions that allow proteins to remain stable, and microorganisms viable, under a wide range of environmental conditions. The increasing evidence that pressure can extend the stable temperature range for enzymes should also expand the use of biocatalysts in the field of high-pressure bioorganic synthesis, among other applications.

We are investigating structure-function relationships associated with the thermo-barophilic behavior of native and recombinant enzymes, e.g., glutamate dehydrogenases (GDHs) from the hyperthermophiles Pyrococcus furiosus and Thermococcus litoralis. Based on a comparison of the two crystal structures, site-directed mutants of the less stable T. litoralis GDH were created to resemble the more thermostable P. furiosus GDH, illustrating the importance of electrostatic interactions in the greater stability of the latter enzyme. Also of interest are structural features (e.g., compressible voids) and solvent effects (e.g., preferential hydration) that contribute to pressure stabilization of GDH at extreme temperatures [1]. The information gained from pressure-temperature studies is being applied toward the development of rational guidelines to alter the stability and pressure response of GDH and other proteins.

Another enzyme system under study is a hyperthermophilic proteasome from the deep-sea methanogen Methanococcus jannaschii. We have cloned the 20S proteasome from M. jannaschii into E. coli and are comparing its properties to those of a less thermophilic recombinant proteasome from Thermoplasma acidophilum. These studies aim to elucidate structural determinants of extreme protein stability, as well as the mechanism(s) by which the M. jannnaschii enzyme is activated and stabilized by pressure at temperatures up to 130°C [2]. The recombinant proteasome from Thermoplasma acidophilum is also the subject of directed evolution experiments, with the goal of expanding its function over a wider range of temperatures.

[1] Sun, M.M.C., Tolliday, N., Vetriani, C., Robb, F.T., Clark, D.S., Protein Sci., 8, 1056-1063, 1999.

[2] Michels, P.C., Clark, D.S., Appl. Environ. Microbiol., 63, 3985-3991, 1997.