The Crystal Structure of D-Glyceraldehyde-3- phosphate Dehydrogenase from the Hyperthermophilic Archaeon Methanothermus fervidus in the Presence of NADP  at 2.1 A Ê Resolution C. Charron 1 , F. Talfournier 2 , M.N. Isupov 3 , J.A. Littlechild 3 , G. Branlant 2 B. Vitoux 1 * and A. Aubry 1 * 1 Laboratoire de Cristallographie et Mode Âlisation des Mate Âriaux Mine Âraux et Biologiques - Groupe Biocristallographie - UPRESA CNRS 7036, Universite  Henri Poincare  Nancy I, BP 239 54506 Vandoeuvre-le Ás-Nancy France 2 Maturation des ARN et Enzymologie Mole Âculaire - UMR CNRS 7567, Universite  Henri Poincare Â, Nancy I, BP 239, 54506, Vandoeuvre-le Ás- Nancy, France 3 Schools of Chemistry and Biological Sciences, University of Exeter, Stocker Road, Exeter EX4 4QD, England The crystal structure of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the archaeon Methanothermus fervidus has been solved in the holo form at 2.1 A Ê resolution by molecular replacement. Unlike bac- terial and eukaryotic homologous enzymes which are strictly NAD  - dependent, GAPDH from this organism exhibits a dual-cofactor speci- ®city, with a marked preference for NADP  over NAD  . The present structure is the ®rst archaeal GAPDH crystallized with NADP  . GAPDH from M. fervidus adopts a homotetrameric quaternary structure which is topologically similar to that observed for its bacterial and eukaryotic counterparts. Within the cofactor-binding site, the positively charged side-chain of Lys33 decisively contributes to NADP  recognition through a tight electrostatic interaction with the adenosine 2 0 -phosphate group. Like other GAPDHs, GAPDH from archaeal sources binds the nicotin- amide moiety of NADP  in a syn conformation with respect to the adja- cent ribose and so belongs to the B-stereospeci®c class of oxidoreductases. Stabilization of the syn conformation is principally achieved through hydrogen bonding of the carboxamide group with the side-chain of Asp171, a structural feature clearly different from what is observed in all presently known GAPDHs from bacteria and eukaryotes. Within the catalytic site, the reported crystal structure de®nitively con- ®rms the essential role previously assigned to Cys140 by site-directed mutagenesis studies. In conjunction with new mutation results reported in this paper, inspection of the crystal structure gives reliable evidence for the direct implication of the side-chain of His219 in the catalytic mechanism. M. fervidus grows optimally at 84  C with a maximal growth temperature of 97  C. The paper includes a detailed comparison of the present structure with four other homologous enzymes extracted from mesophilic as well as thermophilic organisms. Among the various phenomena related to protein thermostabilization, reinforcement of elec- trostatic and hydrophobic interactions as well as a more ef®cient molecu- lar packing appear to be essentially promoted by the occurrence of two additional a-helices in the archaeal GAPDHs. The ®rst one, named a4, is located in the catalytic domain and participates in the enzyme architec- ture at the quaternary structural level. The second one, named aJ, occurs at the C terminus and contributes to the molecular packing within each monomer by ®lling a peripherical pocket in the tetrameric assembly. # 2000 Academic Press Keywords: glyceraldehyde-3-phosphate dehydrogenase; NADP; crystal structure; Archaea; Methanothermus fervidus *Corresponding authors E-mail addresses of the corresponding authors: vitoux@lcm3b.u-nancy.fr; aubry@lcm3b.u-nancy.fr Abbreviations used: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; D-G3P, D-glyceraldehyde-3-phosphate; rmsd, root-mean-square deviation; T  opt, optimal temperature of growth. doi:10.1006/jmbi.2000.3565 available online at http://www.idealibrary.com on J. Mol. Biol. (2000) 297, 481±500 0022-2836/00/010481±20 $35.00/0 # 2000 Academic Press