Investigation of the Structural Basis of Enzyme Hyperthermophilicity
Michael A ARNOTT*, David W HOUGH, and Michael J DANSON
Centre for Extremophile Research, Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath, BA2 7AY, UK
The ability of enzymes to withstand extreme temperatures and remain active is of considerable commercial interest. In seeking to understand the structural basis of enzyme thermostability, many studies have compared enzymes from mesophilic and thermophilic hosts. This study involves the structural comparison of the dimeric citrate synthases (CS) from a thermophile [Thermoplasma acidophilum, (Tp), optimum growth temperature 55｡?, and a hyperthermophile [Pyrococcus furiosus, (Pj), optimum growth temperature 100｡?.
Crystal structures have been determined for CSs from Tp and Pf (1,2). Each monomer consists of a large and a small domain, with the dimeric intersubunit contacts being formed mainly by interactions of the large domain, whereas the small domain is principally concerned with the active site regions. In this study chimeric CSs have been constructed where the small domains have been swapped between the Tp and P fenzymes. The mutated genes have been sequenced and over-expressed, and the recombinant CSs have been characterized. Preliminary results show that the Km values for the substrates oxaloacetate, and acetyl CoA lie between 1 and 10 μM, and the catalytic activities are related to the nature of the small domain. On the other hand, denaturation studies indicate that the structural features responsible for hyperthermophilicity lie principally within the large domain, and that interactions at the subunit interface are crucial for maintaining stability.
The data are discussed with respect to the crystal structures of the enzymes, and the construction of additional mutants to confirm these conclusions is underway.
1. Russell, R.J.M., Hough, D.W., Danson, M.J., and Taylor, G.L. (1994) Structure, 2, 157-1167.
2. Russell, R.J.M, Ferguson, M.C., Hough, D.W., Danson, M.J, and Taylor, G.L. (1997) Biochemistry, 36, 9983-9994.
