Structural Basis of allosteric regulation and substrate specificity of the non-phosphorylating glyceraldehyde 3-Phosphate dehydrogenase from Thermoproteus tenax.
The non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) of the hyperthermophilic Archaeum Thermoproteus tenax is a member of the superfamily of aldehyde dehydrogenases (ALDH). GAPN catalyses the irreversible oxidation of glyceraldehyde 3-phosphate (GAP) to 3-phosphoglycerate in the modified glycolytic pathway of this organism. In contrast to other members of the ALDH superfamily, GAPN from T.tenax (Tt-GAPN) is regulated by a number of intermediates and metabolites. In the NAD-dependent oxidation of GAP, glucose 1-phosphate, fructose 6-phosphate, AMP and ADP increase the affinity for the cosubstrate, whereas ATP, NADP, NADPH and NADH decrease it leaving, however, the catalytic rate virtually unaltered. As we show here, the enzyme also uses NADP as a cosubstrate, displaying, however, unusual discontinuous saturation kinetics indicating different cosubstrate affinities and/or reactivities of the four active sites of the protein tetramer caused by cooperative effects. Furthermore, in the NADP-dependent reaction the presence of activators decreases the overall S0.5 and increases Vmax by a factor of 3. To explore the structural basis for the different effects of both pyridine nucleotides we solved the crystal structure of Tt-GAPN in complex with NAD at 2.2 A resolution and compared it to the binary Tt-GAPN-NADPH structure. Although both pyridine nucleotides show a similar binding mode, NADPH appears to be more tightly bound to the protein via the 2' phosphate moiety. Moreover, we present four co-crystal structures with the activating molecules glucose 1-phosphate, fructose 6-phosphate, AMP and ADP determined at resolutions ranging from 2.3 A to 2.6 A. These crystal structures reveal a common regulatory site able to accommodate the different activators. A phosphate-binding pocket serves as an anchor point ensuring similar binding geometry. The observed conformational changes upon activator binding are discussed in terms of allosteric regulation. Furthermore, we present a crystal structure of Tt-GAPN in complex with the substrate D-GAP at 2.3 A resolution, which allows us to analyse the structural basis for substrate binding, the mechanism of catalysis as well as the stereoselectivity of the enzymatic reaction.
Lorentzen
Esben
E
European Molecular Biology Laboratory, Hamburg Outstation, Notkestr. 85, D-22603 Hamburg, Germany.
Hensel
Reinhard
R
Knura
Thomas
T
Ahmed
Hatim
H
Pohl
Ehmke
E
D000444:Aldehyde Dehydrogenase / Q000737:chemistry*; D000495:Allosteric Site; D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D019992:Databases as Topic; D004305:Dose-Response Relationship, Drug; D004583:Electrons; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) / Q000737:chemistry*; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009249:NADP / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism; D013379:Substrate Specificity; D041542:Thermoproteus / Q000201:enzymology*
D009249:NADP; D000444:Aldehyde Dehydrogenase; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+)