The Crystal Structure of Glyceraldehyde-3-phosphate Dehydrogenase from the Hyperthermophilic Archaeon Methanothermus fervidus in the Presence of NADP at 2.1 Aa Resolution

• Published in: Journal of molecular biology Vol. 297; no. 2; pp. 481 - 500
• Main Authors: Charron, C, Talfournier, F, Isupov, M, Littlechild, J, Branlant, G, Vitoux, B, Aubry, A
• Format: Journal Article
• Language: English
• Published: 24-03-2000
• Subjects: glyceraldehyde-3-phosphate dehydrogenase; hyperthermophilic archaea; Methanothermus fervidus; NADP
• ISSN: 0022-2836
• DOI: 10.1006/jmbi.2000.3565

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 Aa resolution by molecular replacement. Unlike bacterial and eukaryotic homologous enzymes which are strictly NAD super(+)-dependent, GAPDH from this organism exhibits a dual-cofactor specificity, with a marked preference for NADP super(+) over NAD super(+). The present structure is the first archaeal GAPDH crystallized with NADP super(+). 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 super(+) recognition through a tight electrostatic interaction with the adenosine 2'-phosphate group. Like other GAPDHs, GAPDH from archaeal sources binds the nicotinamide moiety of NADP super(+) in a syn conformation with respect to the adjacent ribose and so belongs to the B-stereospecific 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 definitively confirms 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 degree C with a maximal growth temperature of 97 degree 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 electrostatic and hydrophobic interactions as well as a more efficient molecular packing appear to be essentially promoted by the occurrence of two additional alpha -helices in the archaeal GAPDHs. The first one, named alpha 4, is located in the catalytic domain and participates in the enzyme architecture at the quaternary structural level. The second one, named alpha J, occurs at the C terminus and contributes to the molecular packing within each monomer by filling a peripherical pocket in the tetrameric assembly.