From Cold-activity to Hyperstability: the Structural Basis of Enzyme Function at Extreme Temperatures
Michael J. DANSON*, Ursula GERIKE, David W. HOUGH, Rupert J.M. RUSSELL, and Garry L. TAYLOR
Centre for Extremophile Research, Department of Biology and Biochemistry, University of Bath, Bath, BA2 7AY, UK
Enzymes from organisms growing in extreme environments have considerable biotechnological potential, necessitating the understanding of the structural basis of their function and stability under these extraordinary conditions. We have chosen as a model protein the enzyme citrate synthase, and have cloned, sequenced and expressed the gene encoding citrate synthase from Pyrococcus furiosus (optimum growth at 100℃), Sulfolobus solfataricus (80℃), Thermoplasma acidophilum (55℃) and a psychrophilic bacterium, DS2-3R (15℃). The recombinant enzymes all have activities and stabilities commensurate with the growth,optima of their source organisms, and provide a series of homologous proteins for structural studies (1,2).
We will report the crystal structures that we have determined of the dimeric citrate synthases from P. furiosus (3), T. acidophilum (4) and DS2-3R, that from pig (37℃) being already available. These data will be combined with site-directed mutagenesis experiments and protein unfolding studies using differential scanning calorimetry, both of which indicate that subunit dissociation precedes polypeptide unfolding and therefore that the strength of the subunit interactions may determine overall thermostability. Consistent with this, we observe that inter-subunit networks of ionic interactions are unique to the dimer interface of the hyperthermostable citrate synthase, concomitant with a greater inter-subunit surface complementarity. Factors that might stabilise the individual subunits include a more compact folding structure, achieved through fewer and smaller cavities, a higher percentage of buried residues, and shorter loop regions than the mesophilic counterparts.
With respect to enzymic activity at low temperatures, the psychrophilic citrate synthase is a more flexible protein than its thermophilic homologues. This is achieved through extended surface loops, the deletion of proline residues in loops, fewer ionic interactions at the subunit interface, and an increased exposure of hydrophobic residues to solvent. Promotion of high catalytic rates at low temperatures appears to be promoted by a larger entrance to the active site and the electrostatic channelling of the substrates to this site via a polarized surface potential on the enzyme.
1. Muir, J.M., Russell, R.J.M., Hough, D.W., and Danson, M.J. (1995) Citrate synthase from the hyperthermophilic Archaeon Pyrococcusfuriosus. Protein Eng., 8, 583-592.
2. Gerike, U., Russell, N.J., Danson, M.J., and Hough, D.W. (1997) Sequencing and expression of the gene encoding a cold-active citrate synthase from an Antarctic Bacterium, Strain DS2-3R. Eur. J. Biochem. 248, 49-57.
3. Russell, R.J.M., Campbell-Ferguson, J.M., Hough, D.W., Danson, M.J., and Taylor, G.L. (1997) The crystal structure of citrate synthase from the hyperthermophilic Archaeon Pyrococcus furiosus at 1.9A resolution. Biochemistry, USA, 36, 9983-9994.
4. Russell, R.J.M., Hough, D.W., Danson, M.J., and Taylor, G.L. (1994) The crystal structure of citrate synthase from the thermophilic Archaeon Thermoplasma acidophilum. Structure, 2, 1157-1167.
