THE STRUCTURE OF THE THERMOPHILIC GLUTAMATE DEHYDROGENASE FROM THERMOCOCCUS ANI. C. A. Smith, G. E. Norris, E. N. Baker, Department of Biochemistry, Massey University, Palmerston North, New Zealand

How thermostable enzymes maintain their structural integrity and functionality at elevated temperatures (> 90[[ring]]C) remains a key question in structural biology today. In addition to their stability at high temperatures, these heat-stable enzymes generally exhibit less flexibility at lower temperatures and are much less likely to be denatured compared with mesophilic enzymes. These properties make thermostable enzymes potentially important in industry and medicine, and the ability to be able to predict which features are the most important for thermostability will enable important enzymes to be engineered for higher stability. To this end, we have undertaken the structural analysis of the highly thermostable glutamate dehydrogenase (GDH) (t_1/2 at 90[[ring]]C = 12.5 h) from the thermo-philic sulfur-reducing archaeon Thermococcus ANI. GDH catalyses the oxidative deamination of L-glutamate to 2-oxyglutarate and ammonia, and provides an important link between carbon and nitrogen metabolism.

The Thermococcus GDH has been crystallized in two orthorhombic forms; form A, a primitive cell with dimensions 155.3 x 115.1 x 173.4Å, and form B, a C centred cell with dimensions 97.6 x 247.5 x 146.4Å. Two data sets have been collected from form A crystals using an in-house rotating anode (to 3.0Å) and a synchrotron source (to 2.4Å). The V_M was 2.75 ų/Da, assuming 6 monomers (M_r 47,000 Da) per asymmetric unit.

The structure was solved by automated molecular replacement using AMoRe, with a search model derived from the homohexameric Pyrococcus furiosus GDH. The four primitive orthorhombic spacegroups were searched for two independent occurences of one P. furiosus trimer, with the most significant solution observed in P2₁2₁2₁ (correlation 60.2 and R-factor 34.4%). The structure has been refined using molecular dynamics (XPLOR) and restrained least-squares methods (TNT), employing strict 6-fold non-crystallographic symmetry constraints.

The structure consists of two trimers arranged "back-to-back", the interface forming an extended [[beta]]-sheet. The arrangement of each trimer is similar to that observed in the P. furiosus and C. symbiosum structures, and a structural comparison, along with the identification of potential structural determinants of enzyme thermostability will be discussed.