Functional characterization of the phosphorylating D‐glyceraldehyde 3‐phosphate dehydrogenase from the archaeon Methanothermus fervidus by comparative molecular modelling and site‐directed mutagenesis
Authors: Talfournier François , Colloc’h Nathalie , Mornon Jean‐Paul , Branlant Guy 
Published: March 2003

Phosphorylating archaeal d‐glyceraldehyde 3‐phosphate dehydrogenases (GraP‐DHs) share only 15–20% identity with their glycolytic bacterial and eukaryotic counterparts. Unlike the latter which are NAD‐specific, archaeal GraP‐DHs exhibit a dual‐cofactor specificity with a marked preference for NADP. In the present study, we have constructed a three‐dimensional model of the Methanothermus fervidus GraP‐DH based upon the X‐ray structures of the Bacillus stearothermophilus and Escherichia coli GraP‐DHs. The overall structure of the archaeal enzyme is globally similar to homology modelling‐derived structures, in particular for the cofactor binding domain, which might adopt a classical Rossmann fold. M. fervidus GraP‐DH can be considered as a dimer of dimers which exhibits negative and positive cooperativity in binding the coenzymes NAD and NADP, respectively. As expected, the differences between the model and the templates are located mainly within the loops. Based on the predictions derived from molecular modelling, site‐directed mutagenesis was performed to characterize better the cofactor binding pocket and the catalytic domain. The Lys32Ala, Lys32Glu and Lys32Asp mutants led to a drastic increase in the Km value for NADP (i.e. 165‐, 500‐ and 1000‐fold, respectively), thus demonstrating that the invariant Lys32 residue is one of the most important determinants favouring the adenosine 2′‐PO42– binding of NADP. The involvement of the side chain of Asn281, which was postulated to play a role equivalent to that of the Asn313 of bacterial and eukaryotic GraP‐DHs in fixing the position of the nicotinamide ring in a syn orientation [Fabry, S. & Hensel, R. (1988) Gene64, 189–197], was ruled out. Most of the amino acids involved in catalysis and in substrate recognition in bacterial and eukaryotic GraP‐DHs are not conserved in the archaeal enzyme except for the essential Cys149. Inspection of our model suggests that side chains of invariant residues Asn150, Arg176, Arg177 and His210 are located in or near the active site pocket. The Arg177Asn mutation induced strong allosteric properties with the Pi, indicating that this residue should be located near to the intersubunit interfaces. The Arg176Asn mutation led to a 10‐fold decrease in the kcat, a 35‐fold increase in the Km value for d‐glyceraldehyde 3‐phosphate and a 1000‐fold decrease in the acylation rate. These results strongly suggest that Arg176 is involved in the Ps site. The His210Asn mutation increased the pKapp of the catalytic Cys149 from 6.3 to 7.6, although no Cys−/His+ ion pair was detectable [Talfournier, F., Colloc’h, N., Mornon, J.P. & Branlant, G. (1998) Eur. J. Biochem.252, 447–457]. No other invariant amino acid which can play a role as a base catalyst to favour the hydride transfer is located in the active site. The fact that the efficiency of phosphorolysis is 1000‐fold lower when compared to the B. stearothermophilus GraP‐DH suggests significant differences in the nature of the Pi site. Despite these differences, it is likely that the archaeal GraP‐DHs and their bacterial and eukaryotic counterparts have evolved from a common ancestor.