Structure Determination of the Glutamate Dehydrogenase from the Hyperthermophile Thermococcus litoralis and its Comparison with that from Pyrococcus furiosus K. L. Britton 1 , K. S. P. Yip 1 , S. E. Sedelnikova 1 , T. J. Stillman 1 M. W. W. Adams 3 , K. Ma 3 , D. L. Maeder 2 , F. T. Robb 2 , N. Tolliday 2 C. Vetriani 2 , D. W. Rice 1 * and P. J. Baker 1 1 The Krebs Institute for Biomolecular Research Department of Molecular Biology and Biotechnology University of Shef®eld Shef®eld S10 2TN, UK 2 Center of Marine Biotechnology, University of Maryland Biotechnology Institute, 701 E. Pratt Street Baltimore, MD 21202, USA 3 Department of Biochemistry and Molecular Biology University of Georgia, Athens GA 30602, USA Glutamate dehydrogenase catalyses the oxidative deamination of gluta- mate to 2-oxoglutarate with concomitant reduction of NAD(P)  , and has been shown to be widely distributed in nature across species ranging from psychrophiles to hyperthermophiles. Extensive characterisation of this enzyme isolated from hyperthermophilic organisms has led to its adoption as a model system for analysing the determinants of thermal stability. The crystal structure of the extremely thermostable glutamate dehydrogenase from Thermococcus litoralis has been determined at 2.5 A Ê resolution, and has been compared to that from the hyperthermophile Pyrococcus furiosus. The two enzymes are 87 % identical in sequence, yet differ 16-fold in their half-lives at 104  C. This is the ®rst reported com- parative analysis of the structures of a multisubunit enzyme from two closely related yet distinct hyperthermophilies. The less stable T. litoralis enzyme has a decreased number of ion pair interactions; modi®ed patterns of hydrogen bonding resulting from isosteric sequence changes; substitutions that decrease packing ef®ciency; and substitutions which give rise to subtle but distinct shifts in both main-chain and side-chain elements of the structure. This analysis provides a rational basis to test ideas on the factors that confer thermal stability in proteins through a combination of mutagenesis, calorimetry, and structural studies. # 1999 Academic Press Keywords: crystal structure; glutamate dehydrogenase; protein stability; Thermococcus litoralis; Pyrococcus furiosus *Corresponding author Introduction The ability of enzymes from hyperthermophiles to operate for long periods at temperatures in excess of 100  C has prompted much research into the molecular mechanisms that give rise to such remarkable properties (Flam, 1994). Analysis of the structure of hyperthermophilic proteins has high- lighted a range of differences between these enzymes and their mesophilic counterparts, includ- ing increased numbers of ion pairs, reduction in the size of loops and in the number of cavities, reduced ratio of surface area to volume, introduc- tion of additional proline residues, increased hydrophobic interaction at subunit interfaces, increase in the extent of secondary structure for- mation and truncated N and C termini (Elcock, 1998; Vogt et al., 1997; Tanner et al., 1996; Starich et al., 1996; Yip et al., 1995; Chan et al., 1995; Korndo È rfer et al., 1995; Russell et al., 1994). Some of these hypotheses have been tested by mutagenesis and recent work has indicated that increments of stability may be achieved in proteins from meso- philes by a variety of mechanisms such as the inclusion of ``rigidifying'' mutations to ¯exible regions of secondary structure and the introduction of charged-charged interactions (Van Den Burg et al., 1998; Bogin et al., 1998; Zhang et al., 1995). E-mail address of the corresponding author: d.rice@shef®eld.ac.uk Abbreviations used: Pf, P. furiosus; Tl, T. litoralis; GluDH, glutamate dehydrogenase; Cs, C. symbiosum. Article No. jmbi.1999.3205 available online at http://www.idealibrary.com on J. Mol. Biol. (1999) 293, 1121±1132 0022-2836/99/451121±12 $30.00/0 # 1999 Academic Press