Crystal structure of two ternary complexes of phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus with NAD and D-glyceraldehyde 3-phosphate

The crystal structure of the phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus was solved in complex with its cofactor, NAD, and its physiological substrate, D-glyceraldehyde 3-phosphate (D-G3P). To isolate a stable ternary complex, the nucleophilic residue of the active site, Cys(149), was substituted with alanine or serine. The C149A and C149S GAPDH ternary complexes were obtained by soaking the crystals of the corresponding binary complexes (enzyme.NAD) in a solution containing G3P. The structures of the two binary and the two ternary complexes are presented. The D-G3P adopts the same conformation in the two ternary complexes. It is bound in a non-covalent way, in the free aldehyde form, its C-3 phosphate group being positioned in the P(s) site and not in the P(i) site. Its C-1 carbonyl oxygen points toward the essential His(176), which supports the role proposed for this residue along the two steps of the catalytic pathway. Arguments are provided that the structures reported here are representative of a productive enzyme.NAD.D-G3P complex in the ground state (Michaelis complex).     

Cooperativity and noncooperativity in the binding of NAD analogues to rabbit muscle glyceraldehyde-3-phosphate dehydrogenase.

Using NAD analogues as ligands, the structural requirements for negative cooperativity in binding to rabbit muscle glyceraldehyde-3-phosphate dehydrogenase were examined. Although the affinity of nicotinamide hypoxanthine dinucleotide is considerably lower than that of NAD+, it also binds to the enzyme with negative cooperatively. Two pairs of nicotinamide hypoxanthine dinucleotide binding sitess were distinguished, one pair having an affinity for the analogue which is 15 times that of the second pair. Negative cooperativity is also found in the Km values for the analogue. Thus modification of the adenine ring of NAD+ to hypoxanthine does not abolish negative cooperativity in coenzyme binding. Adenosine diphosphoribose binding to the same enzyme shows neither positive nor negative cooperativity, indicating that cooperativity apparently requires an intact nicotinamide ring in the coenzyme structure, under the conditions of these experiments. Occupancy of the nicotinamide subsite of the coenzyme binding site is not necessary for half-of-sites reactivity of alkylating or acylating compounds (Levitzki, A. (1974), J. Mol, Biol. 90, 451-458). However, it can be important in the negative cooperativity in ligand binding, as illustrated by adenosine diphosphoribose which fails to exhibit negative cooperativity. Occupancy of the adenine subsite by adenine is important for stabilization of the enzyme against thermal denaturation. Whether the stabilization is due to an altered conformation of the subunits or stabilization of the preexisting structure of the apoenzyme cannot be determined from these studies. However, nicotinamide hypoxanthine dinucleotide does not contribute to enzyme stability although it serves as a substrate and shows negative cooperativity.     

A phosphate-stimulated NAD(P)+-dependent glyceraldehyde-3-phosphate dehydrogenase in Bacillus cereus

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme of central carbon metabolism, was studied in a Bacillus cereus strain isolated from the phosphate layer from Morocco. Enzymatic assays with cell extracts demonstrated that when grown on Luria-Bertani (LB) medium, B. cereus contains a major NAD+-dependent GAPDH activity and only traces of NADP+-dependent activity, but in cells grown on Pi-supplemented LB medium a strong increase of the NADP+-dependent activity, that became predominant, occurs concurrently with a GAPDH protein increase. Our results show that B. cereus possesses two GAPDH activities, namely NAD+- and NADP+-dependent, catalyzed by two enzymes with distinct coenzyme specificity and different phosphate regulation patterns. The finding of a phosphate-stimulated NADP+-dependent GAPDH in B. cereus indicates that this bacterium can modulate its primary carbon metabolism according to phosphate availability.     

Heavy-atom effects associated with methylmercury (II) binding to rabbit glyceraldehyde-3-phosphate dehydrogenase

The complex of CH₃Hg(II) with the accessible cysteines of glyceraldehyde-3-phosphate dehydrogenase (GAPD, EC 1.2.1.12) from rabbit muscle has been studied by phosphorescence and optically detected magnetic resonance (ODMR) spectroscopy. The wavelength dependence of the phosphorescence decay kinetics has also been measured. Comparison of CH₃Hg(II)–GAPD with GAPD by these methods shows that a specific optically resolved tryptophan site of GAPD is perturbed by the interaction with a nearby mercury atom. The perturbation on the luminescence and ODMR properties is typical of an external heavy-atom effect. Based on the x-ray diffraction structure of the lobster enzyme, it is proposed that the heavy-atom effect results from the interaction of tryptophan-310 with CH₃Hg(II) bound to cysteine-281 in the rabbit muscle enzyme.     

Crystal structure of the branching enzyme I (BEI) from Oryza sativa L with implications for catalysis and substrate binding

Starch-branching enzyme catalyzes the cleavage of α-1, 4-linkages and the subsequent transfer of α-1,4 glucan to form an α-1,6 branch point in amylopectin. Sequence analysis of the rice-branching enzyme I (BEI) indicated a modular structure in which the central α-amylase domain is flanked on each side by the N-terminal carbohydrate-binding module 48 and the α-amylase C-domain. We determined the crystal structure of BEI at a resolution of 1.9 ? by molecular replacement using the Escherichia coli glycogen BE as a search model. Despite three modular structures, BEI is roughly ellipsoidal in shape with two globular domains that form a prominent groove which is proposed to serve as the α-polyglucan-binding site. Amino acid residues Asp344 and Glu399, which are postulated to play an essential role in catalysis as a nucleophile and a general acid/base, respectively, are located at a central cleft in the groove. Moreover, structural comparison revealed that in BEI, extended loop structures cause a narrowing of the substrate-binding site, whereas shortened loop structures make a larger space at the corresponding subsite in the Klebsiella pneumoniae pullulanase. This structural difference might be attributed to distinct catalytic reactions, transglycosylation and hydrolysis, respectively, by BEI and pullulanase.     

Purification and properties of NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase from spinach leaves

An NAD⁺-dependent glyceraldehyde-3-phosphate dehydrogenase (d-glyceraldehyde-3-phosphate:NAD⁺ oxidoreductase (phosphorylating), EC. 1.2.1.12) has been purified from spinach leaves as a homogeneous protein of 150 000 daltons.Kinetic constants of 2.5·10⁻⁴ M and 4 · 10⁻⁴ M have been calculated for NAD⁺ and glyceraldehyde 3-phosphate, respectively.The amino acid composition is characterized by a cysteine content higher than that found in analogous enzymes.On sodium dodecyl sulphate gel electrophoresis, the native enzyme dissociates into two subunits of 37 000 and 14 000 daltons. The two subunits have been isolated in equimolar amounts by gel filtration; end-group analysis shows that alanine is the N-terminal residue of the large subunit, while serine is found at the N-terminus of the small subunit.Comparison of amino acid analyses and peptide maps shows that the two subunits have a different amino acid sequence. These results indicate that the NAD⁺-dependent glyceraldehyde-3-phosphate dehydrogenase, isolated from spinach leaves has an atypical oligomeric structure, the protomer being formed by two different subunits.     

The crystal and solution structures of glyceraldehyde-3-phosphate dehydrogenase reveal different quaternary structures

The presence of an isoform of glyceraldehyde-3-phosphate dehydrogenase (kmGAPDH1p) associated with the cell wall of a flocculent strain of Kluyveromyces marxianus was the first report of a non-cytosolic localization of a glycolytic enzyme, but the mechanism by which the protein is transported to the cell surface is not known. To identify structural features that could account for the multiple localizations of the protein, the three-dimensional structure of kmGAPDH1p was determined by x-ray crystallography and small angle x-ray scattering. The x-ray crystallographic structure of kmGAPDH1p revealed a dimer, although all GAPDH homologs studied thus far have a tetrameric structure with 222 symmetry. Interestingly, the structure of kmGAPDH1p in solution revealed a tetramer with a 70 degrees tilt angle between the dimers. Moreover, the separation between the centers of the dimers composing the kmGAPDH1p tetramer diminished from 34 to 30 A upon NAD(+) binding, this latter value being similar to the observed in the crystallographic models of GAPDH homologs. The less compact structure of apo-kmGAPDH1p could already be the first image of the transition intermediate between the tetramer observed in solution and the dimeric form found in the crystal structure, which we postulate to exist in vivo because of the protein's multiple subcellular localizations in this yeast species.     

Crystal structures of human glycerol 3-phosphate dehydrogenase 1 (GPD1)

Homo sapiens L-alpha-glycerol-3-phosphate dehydrogenase 1 (GPD1) catalyzes the reversible biological conversion of dihydroxyacetone (DHAP) to glycerol-3-phosphate. The GPD1 protein was expressed in Escherichia coli, and purified as a fusion protein with glutathione S-transferase. Here we report the apoenzyme structure of GPD1 determined by multiwavelength anomalous diffraction phasing, and other complex structures with small molecules (NAD+ and DHAP) by the molecular replacement method. This enzyme structure is organized into two distinct domains, the N-terminal eight-stranded beta-sheet sandwich domain and the C-terminal helical substrate-binding domain. An electrophilic catalytic mechanism by the epsilon-NH3+ group of Lys204 is proposed on the basis of the structural analyses. In addition, the inhibitory effects of zinc and sulfate on GPDHs are assayed and discussed.     

Purification and properties of NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase from spinach leaves.

An NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC. 1.2.1.12) has been purified from spinach leaves as a homogeneous protein of 150,000 daltons. Kinetic constants of 2.5 . 10(-4) M and 4 . 10(-4) M have been calculated for NAD+ and glyceraldehyde-3-phosphate, respectively. The amino acid composition is characterized by a cysteine content higher than that found in analogous enzymes. On sodium dodecyl sulphate gel electrophoresis, the native enzyme dissociates into two subunits of 37,000 and 14,000 daltons. The two subunits have been isolated in equimolar amounts by gel filtration; end-group analysis shows that alanine is the N-terminal residue of the large subunit, while serine is found at the N-terminus of the small subunit. Comparison of amino acid analysies and peptide maps shows that the two subunits have a different amino acid sequence. These results indicate that the NAD+-dependent glyceraldehyde-3-phosphate, dehydrogenase, isolated from spinach leaves has an atypical oligomeric structure, the protomer being formed by two different subunits.     

Factors affecting coenzyme binding and subunit interactions in glyceraldehyde-3-phosphate dehydrogenase.

There is no evidence, at pH 9.4, of negative cooperativity in the binding of NAD+ or NADH to rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phorphorylating), EC 1.2.1.12) nor in the binding of acetyl pyridine adenine dinucleotide at pH 7.6 and ph 9.4. The binding of NAD+ to carboxymethylated enzyme at pH 7.6 and pH 9.4 also occurs without cooperativity. The possible implications of these findings for the involvement of ionising groups in the enzyme in the subunit interactions responsible for negative cooperativity, previously reported for coenzyme binding at pH 7.4--8.6, are discussed.     

Differential binding of NAD+ to acyl glyceraldehyde-3-phosphate dehydrogenase and its role in the acyl group transfer reaction.

Enzyme protein fluorescence of di-furylacryloyl-glyceraldehyde-3-phosphate dehydrogenase (di-FA-GPDH:lambda max.excitation 290 nm, lambda max.emission 338 nm) is quenched about 28% on saturation with NAD+. Results of fluorometric titration of di-FA-GPDH with NAD+ suggest the presence of two tight and two loose coenzyme binding sites (Kdiss. 0.1 and 6.0 microM, respectively). Initial rates of the NAD(+)-dependent reaction of di-FA-GPDH with arsenate and phosphate and of mono-FA-GPDH with phosphate have been determined at varying coenzyme concentrations. The data suggest that binding of NAD+ at the tight sites does not activate the acyl group for its reaction with the acceptor (phosphate or arsenate). The group transfer reaction is dependent only on NAD+ binding to the loose sites, which carry the acyl group. The large difference in the NAD+ binding affinity to the two types of sites and their different effects on the group transfer reaction impart a sigmoidal shape to the rate versus [NAD+] plots. The sigmoidicity is abolished if the reactive SH groups at the unacylated sites are blocked by carboxymethylation.     

Crystal structure of the non-regulatory A(4 )isoform of spinach chloroplast glyceraldehyde-3-phosphate dehydrogenase complexed with NADP

Here, we report the first crystal structure of a photosynthetic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) complexed with NADP. The enzyme, purified from spinach chloroplasts, is constituted of a single type of subunit (A) arranged in homotetramers. It shows non-regulated NADP-dependent and NAD-dependent activities, with a preference for NADP. The structure has been solved to 3.0 A resolution by molecular replacement. The crystals belong to space group C222 with three monomers in the asymmetric unit. One of the three monomers generates a tetramer using the space group 222 point symmetry and a very similar tetramer is generated by the other two monomers, related by a non-crystallographic symmetry, using a crystallographic 2-fold axis.The protein reveals a large structural homology with known GAPDHs both in the cofactor-binding domain and in regions of the catalytic domain. Like all other GAPDHs investigated so far, the A(4)-GAPDH belongs to the Rossmann fold family of dehydrogenases. However, unlike most dehydrogenases of this family, the adenosine 2'-phosphate group of NADP does not form a salt-bridge with any positively charged residue in its surroundings, being instead set in place by hydrogen bonds with a threonine residue belonging to the Rossmann fold and a serine residue located in the S-loop of a symmetry-related monomer. While increasing our knowledge of an important photosynthetic enzyme, these results contribute to a general understanding of NADP versus NAD recognition in pyridine nucleotide-dependent enzymes.Although the overall structure of A(4)-GAPDH is similar to that of the cytosolic GAPDH from bacteria and eukaryotes, the chloroplast tetramer is peculiar, in that it can actually be considered a dimer of dimers, since monomers are bound in pairs by a disulphide bridge formed across Cys200 residues. This bridge is not found in other cytosolic or chloroplast GAPDHs from animals, bacteria, or plants other than spinach.     

The NAD+ precursors, nicotinic acid and nicotinamide upregulate glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphate dehydrogenase mRNA in Jurkat cells

To better understand the role of nicotinic acid and nicotinamide in the regulation of the oxidative stress response, we measured the levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and glucose-6-phosphate dehydrogenase (G6PD) mRNA in Jurkat cells treated with these NAD+ precursors. We used a modified nonradioactive Northern blot method and detected the mRNA using 18-mer digoxigenin (DIG)-labeled oligonucleotides as probes. We observed increased levels of the mRNAs for the two enzymes in treated cells. Our findings suggest that the NAD+ precursors may protect against oxidative stress and DNA damage by up-regulating the stress response genes GAPDH and G6PD.     

Dimers generated from tetrameric phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus are inactive but exhibit cooperativity in NAD binding

Tetrameric phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus has been described as a "dimer of dimers" with three nonequivalent interfaces, P-axis (between subunits O and P and between subunits Q and R), Q-axis (between subunits O and Q and between subunits P and R), and R-axis interface (between subunits O and R and between subunits P and Q). O-P dimers, the most stable and the easiest to generate, have been created by selective disruption of hydrogen bonds across the R- and Q-axis interfaces by site-directed mutagenesis. Asp-186 and Ser-48, and Glu-276 and Tyr-46, which are hydrogen bond partners across the R- and Q-axis interfaces, respectively, have been replaced with glycine residues. All mutated residues are highly conserved among GAPDHs from different species and are located in loops. Both double mutants D186G/E276G and Y46G/S48G were dimeric, while all single mutants remained tetrameric. As previously described [Clermont, S., Corbier, C., Mely, Y., Gerard, D., Wonacott, A., and Branlant, G. (1993) Biochemistry 32, 10178-10184], NAD binding to wild type GAPDH (wtGAPDH) was interpreted according to the induced-fit model and exhibited negative cooperativity. However, NAD binding to wtGAPDH can be adequately described in terms of two independent dimers with two interacting binding sites in each dimer. Single mutants D186G, E276G, and Y46G exhibited behavior in NAD binding similar to that of the wild type, while both dimeric mutants D186G/E276G and Y46G/S48G exhibited positive cooperativity in binding the coenzyme NAD. The fact that O-P dimer mutants retained cooperative behavior shows that (1) the P-axis interface is important in transmitting the information induced upon NAD binding inside the O-P dimer from one subunit to the other and (2) the S-loop of the R-axis-related subunit is not directly involved in cooperative binding of NAD in the O-P dimer. In both O-P dimer mutants, the absorption band of the binary enzyme-NAD complex had a highly decreased intensity compared to that of the wild type and, in addition, totally disappeared in the presence of G3P or 1,3-dPG. However, no enzymatic activity was detected, indicating that the formed ternary enzyme-NAD-G3P or -1, 3-dPG complex was not catalytically efficient. In the O-P dimers, the interaction with the S-loop of the R-axis-related subunit is disrupted, and therefore, the S-loop should be less structured. This resulted in increased accessibility of the active site to the solvent, particularly for the adenosine-binding site of NAD. Thus, together, this is likely to explain both the lowered affinity of the dimeric enzyme for NAD and the absence of activity.     

The inhibition of glyceraldehyde-3-phosphate dehydrogenase by nitroxyl (HNO)

Nitroxyl (HNO) has received recent and significant interest due to its novel and potentially important pharmacology. However, the chemical/biochemical mechanism(s) responsible for its biological activity remain to be established. Some of the most important biological targets for HNO are thiols and thiol proteins. Consistent with this, it was recently reported that HNO inhibits the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a protein with a catalytically important cysteine thiol at its active site. Interestingly, it was reported that intracellular GAPDH inhibition occurred without significantly altering the cellular thiol redox status of glutathione. Herein, the nature of this reaction specificity was examined. HNO is found to irreversibly inhibit GAPDH in a manner that can be protected against by one of its substrates, glyceraldehyde-3-phosphate (G-3-P). These results are consistent with the idea that HNO has the ability to react with and oxidize a variety of intracellular thiols and the ease or facility of cellular re-reduction of the thiol targets can determine the target specificity.     

Mapping of the interaction site of CP12 with glyceraldehyde-3-phosphate dehydrogenase from Chlamydomonas reinhardtii. Functional consequences for glyceraldehyde-3-phosphate dehydrogenase

The 8.5 kDa chloroplast protein CP12 is essential for assembly of the phosphoribulokinase/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) complex from Chlamydomonas reinhardtii. After reduction of this complex with thioredoxin, phosphoribulokinase is released but CP12 remains tightly associated with GAPDH and downregulates its NADPH-dependent activity. We show that only incubation with reduced thioredoxin and the GAPDH substrate 1,3-bisphosphoglycerate leads to dissociation of the GAPDH/CP12 complex. Consequently, a significant twofold increase in the NADPH-dependent activity of GAPDH was observed. 1,3-Bisphosphoglycerate or reduced thioredoxin alone weaken the association, causing a smaller increase in GAPDH activity. CP12 thus behaves as a negative regulator of GAPDH activity. A mutant lacking the C-terminal disulfide bridge is unable to interact with GAPDH, whereas absence of the N-terminal disulfide bridge does not prevent the association with GAPDH. Trypsin-protection experiments indicated that GAPDH may be also bound to the central alpha-helix of CP12 which includes residues at position 36 (D) and 39 (E). Mutants of CP12 (D36A, E39A and E39K) but not D36K, reconstituted the GAPDH/CP12 complex. Although the dissociation constants measured by surface plasmon resonance were 2.5-75-fold higher with these mutants than with wild-type CP12 and GAPDH, they remained low. For the D36K mutation, we calculated a 7 kcal.mol(-1) destabilizing effect, which may correspond to loss of the stabilizing effect of an ionic bond for the interaction between GAPDH and CP12. It thus suggests that electrostatic forces are responsible for the interaction between GAPDH and CP12.