The crystal structure of D-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaeon Methanothermus fervidus in the presence of NADP(+) at 2.1 angstrom resolution

The crystal structure of the glyceraldehyde-3-phosphate dehydrogenase (GAPD H) from the archaeon Methanothermus fervidus has been solved in the hole fo rm at 2.1 Angstrom resolution by molecular replacement. Unlike bacterial an d eukaryotic homologous enzymes which are strictly NAD(+)-dependent, GAPDH from this organism exhibits a dual-cofactor specificity, with a marked pref erence for NADP(+) over NAD(+). The present structure is the first archaeal GAPDH crystallized with NADP(+). GAPDH from M. fervidus adopts a homotetra meric 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 aden osine 2'-phosphate group. Like other GAPDHs, GAPDH from archaeal sources bi nds the nicotinamide moiety of NADP+ in a syn conformation with respect to the adjacent ribose and so belongs to the B-stereospecific class of oxidore ductases. Stabilization of the syn conformation is principally achieved thr ough hydrogen bonding of the carboxamide group with the side-chain of Asp17 1, a structural feature clearly different from what is observed in all pres ently known GAPDHs from bacteria and eukaryotes. Within the catalytic site, the reported crystal structure definitively confirms the essential role pr eviously assigned to Cys140 by site-directed mutagenesis studies. In conjun ction with new mutation results reported in this paper, inspection of the c rystal structure gives reliable evidence for the direct implication of the side-chain of His219 in the catalytic mechanism. M. fervidus grows optimall y at 84 degrees C with a maximal growth temperature of 97 degrees C. The pa per includes a detailed comparison of the present structure with four other homologous enzymes extracted from mesophilic as well as thermophilic organ isms. Among the various phenomena related to protein thermostabilization, r einforcement of electrostatic and hydrophobic interactions as well as a mor e efficient molecular packing appear to be essentially promoted by the occu rrence of two additional alpha-helices in the archaeal GAPDHs. The first on e, named alpha 4, is located in the catalytic domain and participates in th e enzyme architecture at the quaternary structural level. The second one, n amed alpha J, occurs at the C terminus and contributes to the molecular pac king within each monomer by filling a peripherical pocket in the tetrameric assembly. (C) 2000 Academic Press.