Extremophiles are microorganisms growing in environmental extremes of temperature (-2 to 15°C and 60-110°C), salinity (2-5M NaCl), pH (<4 and >9), and/or pressure (>400 atmospheres). Consequently, their cellular components are remarkably stable entities, and they provide a unique source of hyperstable enzymes: 'Extremozymes'. This lecture will focus on our current understanding of the structure, function and stability of extremozymes, with particular emphasis on their adaptation to extremes of temperature.

What is the structural basis of enzyme stability to extreme temperatures?
The strategies used by extremozymes to achieve stability and activity at both low and high temperatures have been investigated in our laboratory by the determination and analysis of protein 3D-structures from organisms spanning the biological range of temperatures. The dimeric enzyme, citrate synthase, is currently our best-studied system, where we have determined high-resolution structures of a series of homologues from organisms growing optimally at 10, 37, 55, 80 and 100°C. Structural trends have been identified, and these include both adaptations within individual subunits and an apparent strengthening of the inter-subunit contacts. These potential adaptations have been correlated with simulations of protein inter-atomic forces and how these are thought to change with temperature. Possible stabilising features have then been tested by site-directed mutagenesis.

Does thermostability limit catalytic activity?
We are now correlating these structural studies with investigations of enzyme thermoactivity. At their organisms' growth temperatures, the individual citrate synthases have similar catalytic activities, which is surprising given that enzymatic activity increases 1.5-2.5 fold with every 10°C rise in assay temperature, as long as thermal inactivation is not occurring. Quantum mechanical - molecular mechanical simulations, and kinetic and mechanistic studies of the 37°C and 55°C citrate synthases, will be presented to address the relationship of stability and activity, and how these two parameters might be independently manipulated.

[1] Danson, M.J. and Hough, D.W. Structure, function and stability of enzymes from the Archaea, Trends in Microbiology 6, 307-314, 1998.

[2] Hough, D.W. & Danson, M.J. Extremozymes, Curr. Opinion Chem. Biol. 3, 39-46, 1999.