NAD+-dependent Glyceraldehyde-3-phosphate Dehydrogenase from Thermoproteus tenax

Abstract

The hyperthermophilic archaeum Thermoproteus tenax possesses two glyceraldehyde-3-phosphate dehydrogenases differing in cosubstrate specificity and phosphate dependence of the catalyzed reaction. NAD ؉ -dependent glyceraldehyde-3-phosphate dehydrogenase catalyzes the phosphate-independent irreversible oxidation of D-glyceraldehyde 3-phosphate to 3-phosphoglycerate. The coding gene was cloned, sequenced, and expressed in Escherichia coli. Sequence comparisons showed no similarity to phosphorylating glyceraldehyde-3-phosphate dehydrogenases but revealed a relationship to aldehyde dehydrogenases, with the highest similarity to the subgroup of nonphosphorylating glyceraldehyde-3-phosphate dehydrogenases.The activity of the enzyme is affected by a series of metabolites. All effectors tested influence the affinity of the enzyme for its cosubstrate NAD ؉ . Whereas NADP(H), NADH, and ATP reduce the affinity for the cosubstrate, AMP, ADP, glucose 1-phosphate, and fructose 6-phosphate increase the affinity for NAD ؉ . Additionally, most of the effectors investigated induce cooperativity of NAD ؉ binding. The irreversible catabolic oxidation of glyceraldehyde 3-phosphate, the control of the enzyme by energy charge of the cell, and the regulation by intermediates of glycolysis and glucan degradation identify the NAD ؉ -dependent glyceraldehyde-3-phosphate dehydrogenase as an integral constituent of glycolysis in T. tenax. Its regulatory properties substitute for those lacking in the reversible nonregulated pyrophosphate-dependent phosphofructokinase in this variant of the EmbdenMeyerhof-Parnas pathway.

The Ferredoxin-dependent Conversion of Glyceraldehyde-3-phosphate in the Hyperthermophilic ArchaeonPyrococcus furiosus Represents a Novel Site of Glycolytic Regulation

“…Indeed, we have demonstrated that regulation does occur at this level. An analogous situation has recently been described in T. tenax (11). In addition to GAPDH/PGK, this hyperthermophilic archaeon expresses an alternative enzyme that is homologous to the GAPN-type aldehyde dehydrogenase of some Bacteria and photosynthetic Eukarya.…”

“…1 In Bacteria and Eukarya, the enzyme-couple glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 3-phosphoglycerate kinase (PGK) catalyzes the two-step conversion of GAP to 3-phosphoglycerate in a bidirectional manner, i.e., during glycolysis as well as gluconeogenesis. The corresponding enzymes in Archaea, however, have been proposed to be mainly involved in the anabolic gluconeogenesis process (10,11). The glycolytic conversion of GAP in the hyperthermophilic Archaeon Pyrococcus furiosus has recently been reported to be phosphate-independent and is catalyzed by an unprecedented enzyme glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR) (12).…”

The Crystal Structure of the Allosteric Non-phosphorylating Glyceraldehyde-3-phosphate Dehydrogenase from the Hyperthermophilic Archaeum Thermoproteus tenax

“…These enzymes catalyze the irreversible oxidation of aldehydes to their respective carboxylic acids. The highest sequence identities are found for other D-GAP-specific ALDHs from bacteria as well as plants (1). In contrast to these NADP ϩ -dependent enzymes, Tt-GAPN explicitly uses NAD ϩ as cosubstrate and moreover exhibits allosteric properties that have not been described for any other ALDH yet investigated (1).…”

“…All effectors induce positive cooperativity, with NADH showing the highest Hill coefficient of all effectors of 1.9. Due to its allosteric properties, Tt-GAPN plays a crucial role in regulating carbohydrate catabolism, thus enabling the organism to respond immediately to physiological requirements (1). Generally, the activity of GAPN is counteracted by the NADP ϩ -dependent phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which preferentially exerts anabolic functions.…”

“…The non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) 1 from the hyperthermophilic archaeum Thermoproteus tenax (Tt-GAPN) catalyzes the NAD ϩ -mediated phosphate-independent irreversible oxidation of D-glyceraldehyde 3-phosphate (D-GAP) to 3-phosphoglycerate. The enzyme is regulated by a number of effectors that modulate the binding of its cosubstrate NAD ϩ .…”