The crystal structure of the allosteric non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaeum Thermoproteus tenax

The NAD(+)-dependent non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) from the hyperthermophilic archaeum Thermoproteus tenax represents an archaeal member of the diverse superfamily of aldehyde dehydrogenases (ALDHs). GAPN catalyzes the irreversible oxidation of d-glyceraldehyde 3-phosphate to 3-phosphoglycerate. In this study, we present the crystal structure of GAPN in complex with its natural inhibitor NADP(+) determined by multiple anomalous diffraction methods. The structure was refined to a resolution of 2.4 A with an R-factor of 0.21. The overall fold of GAPN is similar to the structures of ALDHs described previously, consisting of three domains: a nucleotide-binding domain, a catalytic domain, and an oligomerization domain. Local differences in the active site are responsible for substrate specificity. The inhibitor NADP(+) binds at an equivalent site to the cosubstrate-binding site of other ALDHs and blocks the enzyme in its inactive state, possibly preventing the transition to the active conformation. Structural comparison between GAPN from the hyperthermophilic T. tenax and homologs of mesophilic organisms establishes several characteristics of thermostabilization. These include protection against heat-induced covalent modifications by reducing and stabilizing labile residues, a decrease in number and volume of empty cavities, an increase in beta-strand content, and a strengthening of subunit contacts by ionic and hydrophobic interactions.     

Characterization of the amino acids involved in substrate specificity of nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans

In order to address the molecular basis of the specificity of aldehyde dehydrogenase for aldehyde substrates, enzymatic characterization of the glyceraldehyde 3-phosphate (G3P) binding site of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) from Streptococcus mutans has been undertaken. In this work, residues Arg-124, Tyr-170, Arg-301, and Arg-459 were changed by site-directed mutagenesis and the catalytic properties of GAPN mutants investigated. Changing Tyr-170 into phenylalanine induces no major effect on k(cat) and K(m) for d-G3P in both acylation and deacylation steps. Substitutions of Arg-124 and Arg-301 by leucine and Arg-459 by isoleucine led to distinct effects on K(m), on k(cat), or on both. The rate-limiting step of the R124L GAPN remains deacylation. Pre-steady-state analysis and substrate isotope measurements show that hydride transfer remains rate-determining in acylation. Only the apparent affinity for d-G3P is decreased in both acylation and deacylation steps. Substitution of Arg-459 by isoleucine leads to a drastic effect on the catalytic efficiency by a factor of 10(5). With this R459L GAPN, the rate-limiting step is prior to hydride transfer, and the K(m) of d-G3P is increased by at least 2 orders of magnitude. Binding of NADP leads to a time-dependent formation of a charge transfer transition at 333 nm between the pyridinium ring of NADP and the thiolate of Cys-302, which is not observed with the holo-wild type. Accessibility of Cys-302 is shown to be strongly decreased within the holostructure. The substitution of Arg-301 by leucine leads to an even more drastic effect with a change of the rate-limiting step similar to that observed for R459I GAPN. Taking into account the three-dimensional structure of GAPN from S. mutans and the data of the present study, it is proposed that 1) Tyr-170 is not essential for the catalytic event, 2) Arg-124 is only involved in stabilizing d-G3P binding via an interaction with the C-3 phosphate, and 3) Arg-301 and Arg-459 participate not only in d-G3P binding via interaction with C-3 phosphate but also in positioning efficiently d-G3P relative to Cys-302 within the ternary complex GAPN.NADP.d-G3P.     

Evidence for the chemical activation of essential cys-302 upon cofactor binding to nonphosphorylating glyceraldehyde 3-phosphate dehydrogenase from Streptococcus mutans.

Nonphosphorylating glyceraldehyde 3-phosphate dehydrogenase (GAPN) from Streptococcus mutans which catalyzes the irreversible oxidation of D-glyceraldehyde-3 phosphate (D-G3P) into 3-phosphoglycerate (3-PGA) in the presence of NADP belongs to the aldehyde dehydrogenase (ALDH) superfamily. Oxidation of D-G3P into 3-PGA by GAPN involves the formation of a covalent enzyme intermediate via the nucleophilic attack of the invariant Cys-302. Titration of Cys-302 in the apo-enzyme by two different kinetic probes, iodoacetamide and 2,2'-dipyridyl disulfide, shows a pK(app) of 8.5 and a chemical reactivity surprisingly low compared to a reactive and accessible thiolate. Binding of NADP causes a strong increase of the reactivity of Cys-302-which is time dependent-with a pK(app) shift from 8.5 to 6.1. Concomitant with the increase in the Cys-302 reactivity, an additional protein fluorescence quenching is observed. These data suggest that cofactor binding induces at least a local conformational rearrangement within the active site. The efficiency of the rearrangement depends on the structure of the cofactors and on the protonation of an amino acid with a pK(app)( )()of 5.7. The rate of the rearrangement also strongly increases when temperature decreases. The data on the conformational rearrangement also reveal an amino acid with a pK(app) of 7.6 whose deprotonation increases the reactivity of the thiolate of Cys-302 by a 3-fold factor. The nature of the amino acid involved-which should be located close to Cys-302 in the holo-active form-is likely the invariant Glu-268. Changing Glu-268 into Ala or Cys-302 into Ala leads to mutants in which the rearrangement is only efficient in the presence of saturating concentrations of both NADP and G3P. The structural aspects of the conformational rearrangement occurring during the catalytic process in the wild-type GAPN should include at least reorientation of both Cys-302 and Glu-268 side chains and repositioning of the nicotinamide ring of the cofactor to permit the chemical activation of Cys-302 and the formation of an efficient ternary complex. Thus, it is likely that the conformation of the active site in the reported X-ray structures of ALDHs determined so far in the presence of cofactor, in which the side chains of Cys-302 and Glu-268 are 6.7 A apart from each other, does not represent the biological active form.     

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.     

Apo and holo crystal structures of an NADP-dependent aldehyde dehydrogenase from Streptococcus mutans.

The aldehyde dehydrogenases (ALDHs) are a superfamily of multimeric enzymes which catalyse the oxidation of a broad range of aldehydes into their corresponding carboxylic acids with the reduction of their cofactor, NAD or NADP, into NADH or NADPH. At present, the only known structures concern NAD-dependent ALDHs. Three structures are available in the Protein Data Bank: two are tetrameric and the other is a dimer. We solved by molecular replacement the first structure of an NADP-dependent ALDH isolated from Streptococcus mutans, in its apo form and holo form in complex with NADP, at 1.8 and 2.6 A resolution, respectively. Although the protein sequence shares only approximately 30 % identity with the other solved tetrameric ALDHs, the structures are very similar. However, a large local conformational change in the region surrounding the 2' phosphate group of the adenosine moiety is observed when the enzyme binds NADP, in contrast to the NAD-dependent ALDHs.Structure and sequence analyses reveal several properties. A small number of residues seem to determine the oligomeric state. Likewise, the nature (charge and volume) of the residue at position 180 (Thr in ALDH from S. mutans) determines the cofactor specificity in comparison with the structures of NAD-dependent ALDHs. The presence of a hydrogen bond network around the cofactor not only allows it to bind to the enzyme but also directs the side-chains in a correct orientation for the catalytic reaction to take place. Moreover, a specific part of this network appears to be important in substrate binding. Since the enzyme oxidises the same substrate, glyceraldehyde-3-phosphate (G3P), as NAD-dependent phosphorylating glyceraldehyde-3-phosphate dehydrogenases (GAPDH), the active site of GAPDH was compared with that of the S. mutans ALDH. It was found that Arg103, Arg283 and Asp440 might be key residues for substrate binding.     

Chemical mechanism and substrate binding sites of NADP-dependent aldehyde dehydrogenase from Streptococcus mutans

Non-phosphorylating glyceraldehyde 3-phosphate dehydrogenase from Streptococcus mutans (GAPN) belongs to the aldehyde dehydrogenase (ALDH) family, which catalyzes the irreversible oxidation of a wide variety of aldehydes into acidic compounds via a two-step mechanism: first, the acylation step involves the formation of a covalent ternary complex ALDH-cofactor-substrate, followed by the oxidoreduction process which yields a thioacyl intermediate and reduced cofactor and second, the rate-limiting deacylation step. Structural and molecular factors involved in the chemical mechanism of GAPN have recently been examined. Specifically, evidence was put forward for the chemical activation of catalytic Cys-302 upon cofactor binding to the enzyme, through a local conformational rearrangement involving the cofactor and Glu-268. In addition, the invariant residue Glu-268 was shown to play an essential role in the activation of the water molecule in the deacylation step. For E268A/Q mutant GAPNs, nucleophilic compounds like hydrazine and hydroxylamine were shown to bind and act as substrates in this step. Further studies were focused at understanding the factors responsible for the stabilization and chemical activation of the covalent intermediates, using X-ray crystallography, site-directed mutagenesis, kinetic and physico-chemical approaches. The results support the involvement of an oxyanion site including the side-chain of Asn-169. Finally, given the strict substrate-specificity of GAPN compared to other ALDHs with wide substrate specificity, one has also initiated the characterization of the G3P binding properties of GAPN. These results will be presented and discussed from the point of view of the evolution of the catalytic mechanisms of ALDH.     

Chemical mechanism and substrate binding sites of NADP-dependent aldehyde dehydrogenase from Streptococcus mutans

Non-phosphorylating glyceraldehyde 3-phosphate dehydrogenase from Streptococcus mutans (GAPN) belongs to the aldehyde dehydrogenase (ALDH) family, which catalyzes the irreversible oxidation of a wide variety of aldehydes into acidic compounds via a two-step mechanism: first, the acylation step involves the formation of a covalent ternary complex ALDH-cofactor-substrate, followed by the oxidoreduction process which yields a thioacyl intermediate and reduced cofactor and second, the rate-limiting deacylation step. Structural and molecular factors involved in the chemical mechanism of GAPN have recently been examined. Specifically, evidence was put forward for the chemical activation of catalytic Cys-302 upon cofactor binding to the enzyme, through a local conformational rearrangement involving the cofactor and Glu-268. In addition, the invariant residue Glu-268 was shown to play an essential role in the activation of the water molecule in the deacylation step. For E268A/Q mutant GAPNs, nucleophilic compounds like hydrazine and hydroxylamine were shown to bind and act as substrates in this step. Further studies were focused at understanding the factors responsible for the stabilization and chemical activation of the covalent intermediates, using X-ray crystallography, site-directed mutagenesis, kinetic and physico-chemical approaches. The results support the involvement of an oxyanion site including the side-chain of Asn-169. Finally, given the strict substrate-specificity of GAPN compared to other ALDHs with wide substrate specificity, one has also initiated the characterization of the G3P binding properties of GAPN. These results will be presented and discussed from the point of view of the evolution of the catalytic mechanisms of ALDH.     

Engineered nonphosphorylating glyceraldehyde 3-phosphate dehydrogenase at position 268 binds hydroxylamine and hydrazine as acyl acceptors.

Nonphosphorylating nicotinamide adenine dinucleotide (phosphate)-dependent aldehyde dehydrogenases (ALDHs) catalyze the oxidation of aldehydes into either nonactivated acids or CoA-activated acids. The NADP-dependent nonphosphorylating glyceraldehyde 3-phosphate dehydrogenase (GAPN) belongs to the first subclass. It catalyzes the irreversible oxidation of glyceraldehyde 3-phosphate into 3-phosphoglycerate via a two step mechanism in which deacylation is rate-limiting. Recent studies on GAPN from Streptococcus mutans have shown that residue Glu268 plays an essential role only in the deacylation step [Marchal, S., Rahuel-Clermont, S. & Branlant, G. (2000) Biochemistry 39, 3327-3335]. The substitution of Glu268 by alanine or glutamine leads to mutants in which the attacking water molecule involved in the hydrolytic process is poorly activated. Activity can be restored by the presence of hydroxylamine and hydrazine. Neutral and protonated forms of both nucleophiles are recognized by the deacylating subsite of both mutants. pH rate profiles of deacylation show pK(a) values of 6.3 and 8.1 with hydroxylamine and hydrazine, respectively, which are those of the nucleophiles in solution. The increase in enzymatic rate is probably due to a high local concentration and not to a change of the chemical reactivity of both nucleophiles upon their binding within the active site of both mutants. The deacylation subsite of the wild-type also binds hydroxylamine and hydrazine but as inhibitors of the hydrolytic process and not as acyl acceptors. Altogether, the results point out the crucial role of the carboxyl group of Glu268 in preventing nucleophiles, other than water, from binding as efficient acyl acceptors. This may also explain why CoA-dependent ALDHs never possesses a glutamate residue at position 268.     

Coenzyme specificity in aldehyde dehydrogenase

Influences on coenzyme preference are explored. Lysine 137 (192 in class 1/2 ALDH) lies close to the adenine ribose, directly interacting with the adenine ribose in NAD-specific ALDHs and the 2′-phosphate of NADP in NADP-specific ALDHs. Lys-137 in class 3 ALDH interacts with the adenine ribose indirectly through an intervening water molecule. However, this residue is present in all ALDHs and, as a result, is unlikely to directly influence coenzyme specificity. Glutamate 140 (195) coordinates the 2′- and 3′-hydroxyls of the adenine ribose of NAD in the class 3 tertiary structure. Thus, it appeared that this residue would influence coenzyme specificity. Mutation to aspartate, asparagine, glutamine or threonine shifts the coenzyme specificity towards NADP, but did not completely change the specificity. Still, the mutants show the 2′-phosphate of NADP is repelled by Glu-140 (195). Although Glu-140 (195) has a major influence on coenzyme specificity, it is not the only influence since class 3 ALDHs, can use both coenzymes, and class 2 ALDHs, which are NAD-specific, have a glutamate at this position. One explanation may be that the larger space between Lys-137 (192) and the adenine ribose hydroxyls in the class 3 ALDH:NAD binary structure may provide space to accommodate the 2′-phosphate of NADP. Also, a structural shift upon binding NADP may also occur in class 3 ALDHs to help accommodate the 2′-phosphate of NADP.     

Covalent binding of 3-pyridinealdehyde nicotinamide adenine dinucleotide and substrate to glyceraldehyde 3-phosphate dehydrogenase.

Glyceraldehyde 3-phosphate dehydrogenase (D-glyceraldehyde-3-phoshate:nicotinamide adenine dinucleotide oxidoreductase (phosphorylating), EC 1.2.1.12) forms a complex with 3-pyridinealdehyde-NAD which survives precipitation with 7% perchloric acid. The molar ratio bound 3-pyridinealdehyde-NAD to the enzyme is 2.5 to 2.9. Lactate, malate, and alcohol dehydrogenases do not form acid-precipitable complexes with 3-pyridinealdehyde-NAD. 3-Pyridinealdehyde-deamino-NAD or glyceraldehyde 3-phosphate also forms an acid-stable complex with glyceraldehyde 3-phosphate dehydrogenase; however, NAD, 3-acetylpyridine-NAD, or thionicotinamide-NAD does not produce an acid-stable complex. Incubation of the glyceraldehyde 3-phosphate dehydrogenase with glyceraldehyde 3-phosphate, acetyl phosphate, iodoacetic acid, or iodosobenzoate inhibits the formation of the acid-stable complex with 3-pyridinealdehyde-NAD. Glyceraldehyde 3-phosphate or 3-pyridinealdehyde-NAD also prevents carboxymethylation of the active site cysteine-149 by[14-C]iodoacetic acid. These studies indicate that the aldehyde group of 3-pyridinealdehyde-NAD forms a thiohemiacetal linkage with cysteine-149 which is the substrate binding site for the dehydrogenase reaction. These findings may account for the fact that 3-pyridinealdehyde-NAD strongly inhibits the dehydrogenase and esterase activities of 3-pyridinealdehyde-NAD forms a thiohemiacetal linkage with cysteine-149 which is the substrate binding site for the dehydrogenase reaction. These findings may account for the fact that 3-pyridinealdehyde-NAD strongly inhibits the dehydrogenase and esterase activities of glyceraldehyde 3-phosphate dehydrogenase which require reduced cysteine-149. However, the analogue does not inhibit the acetyl phosphates activity of the enzyme for which the active site sulfhydryl residues must be oxidized.     

Sequence, expression, and function of the gene for the nonphosphorylating, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase of Streptococcus mutans.

We report the sequencing of a 2,019-bp region of the Streptococcus mutans NG5 genome which contains a 1,428-bp open reading frame (ORF) whose putative translation product had 50% identity to the amino acid sequences of the nonphosphorylating, NADP-dependent glyceraldehyde-3-phosphate dehydrogenases (GAPN) from maize and pea. This ORF is located approximately 200 bp downstream of the ptsI gene coding for enzyme I of the phosphoenolpyruvate:sugar phosphotransferase transport system. Mutant BCH150, in which the putative gapN gene had been inactivated, lacked GAPN activity that was present in the wild-type strain, thus positively identifying the ORF as the S. mutans gapN gene. Another strain of S. mutans, DC10, which contains an insertionally inactivated ptsI gene, still possessed GAPN activity, as did S. salivarius ATCC 25975, which contains an insertion element between the ptsI and gapN genes. Since the wild-type S. mutans NG5 lacks both glucose-6-phosphate dehydrogenase and NADH:NADP oxidoreductase activities, the NADP-dependent glyceraldehyde-3-phosphate dehydrogenase is important as a means of generating NADPH for biosynthetic reactions.     

Poly-β-hydroxybutyrate biosynthesis and the regulation of glucose metabolism in Azotobacter beijerinckii

Azotobacter beijerinckii possesses the enzymes of both the Entner-Doudoroff and the oxidative pentose phosphate cycle pathways of glucose catabolism and both pathways are subject to feedback inhibition by products of glucose oxidation. The allosteric glucose 6-phosphate dehydrogenase utilizes both NADP(+) and NAD(+) as electron acceptors and is inhibited by ATP, ADP, NADH and NADPH. 6-Phosphogluconate dehydrogenase (NADP-specific) is unaffected by adenosine nucleotides but is strongly inhibited by NADH and NADPH. The formation of pyruvate and glyceraldehyde 3-phosphate from 6-phosphogluconate by the action of the Entner-Doudoroff enzymes is inhibited by ATP, citrate, isocitrate and cis-aconitate. Glyceraldehyde 3-phosphate dehydrogenase is unaffected by adenosine and nicotinamide nucleotides but the enzyme is non-specific with respect to NADP and NAD. Citrate synthase is strongly inhibited by NADH and the inhibition is reversed by the addition of AMP. Isocitrate dehydrogenase, a highly active NADP-specific enzyme, is inhibited by NADPH, NADH, ATP and by high concentrations of NADP(+). These findings are discussed in relation to the massive synthesis of poly-beta-hydroxybutyrate that occurs under certain nutritional conditions. We propose that synthesis of this reserve material, to the extent of 70% of the dry weight of the organism, serves as an electron and carbon ;sink' when conditions prevail that would otherwise inhibit nitrogen fixation and growth.     

The activation of glycolysis performed by the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase in the model system

Influence of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) on glycolysis was investigated. The addition of GAPN-which oxidizes glyceraldehyde-3-phosphate directly to the 3-phosphoglyceric acid-led to the strong increase in the rate of lactate accumulation in the rat muscle extract with low ADP content. The lactate accumulation was also observed in the presence of GAPN in rat muscle extract, which contained only ATP and no ADP. This can be the evidence of the "futile cycle" stimulated by GAPN. Here ADP can be regenerated from ATP by the phosphoglycerate kinase reaction. The high resistance of GAPN from Streptococcus mutans towards inactivation by natural oxidant-H(2)O(2) was showed. This feature distinguishes GAPN from phosphorylating glyceraldehyde-3-phosphate dehydrogenase, which is very sensitive to modification by hydrogen peroxide. A possible role of the oxidants and non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase in the regulation of glycolysis is discussed.     

Biochemical and genetic characterization of the three metabolic routes in Thermococcus kodakarensis linking glyceraldehyde 3-phosphate and 3-phosphoglycerate

In the classical Embden-Meyerhof (EM) pathway for glycolysis, the conversion between glyceraldehyde 3-phosphate (GAP) and 3-phosphoglycerate (3-PGA) is reversibly catalysed by phosphorylating GAP dehydrogenase (GAPDH) and phosphoglycerate kinase (PGK). In the Euryarchaeota Thermococcus kodakarensis and Pyrococcus furiosus, an additional gene encoding GAP:ferredoxin oxidoreductase (GAPOR) and a gene similar to non-phosphorylating GAP dehydrogenase (GAPN) are present. In order to determine the physiological roles of the three routes that link GAP and 3-PGA, we individually disrupted the GAPOR, GAPN, GAPDH and PGK genes (gor, gapN, gapDH and pgk respectively) of T. kodakarensis. The Δgor strain displayed no growth under glycolytic conditions, confirming its proposed function to generate reduced ferredoxin for energy generation in glycolysis. Surprisingly, ΔgapN cells also did not grow under glycolytic conditions, suggesting that GAPN plays a key role in providing NADPH under these conditions. Disruption of gor and gapN had no effect on gluconeogenic growth. Growth experiments with the ΔgapDH and Δpgk strains indicated that, unlike their counterparts in the classical EM pathway, GAPDH/PGK play a major role only in gluconeogenesis. Biochemical analyses indicated that T. kodakarensis GAPN did not recognize aldehyde substrates other than d-GAP, preferred NADP(+) as cofactor and was dramatically activated with glucose 1-phosphate.     

The first crystal structure of a thioacylenzyme intermediate in the ALDH family: new coenzyme conformation and relevance to catalysis

Crystal structures of several members of the nonphosphorylating CoA-independent aldehyde dehydrogenase (ALDH) family have shown that the peculiar binding mode of the cofactor to the Rossmann fold results in a conformational flexibility for the nicotinamide moiety of the cofactor. This has been hypothesized to constitute an essential feature of the catalytic mechanism because the conformation of the cofactor required for the acylation step is not appropriate for the deacylation step. In the present study, the structure of a reaction intermediate of the E268A-glyceraldehyde 3-phosphate dehydrogenase (GAPN) from Streptococcus mutans, obtained by soaking the crystals of the enzyme/NADP complex with the natural substrate, is reported. The substrate is bound covalently in the four monomers and presents the geometric characteristics expected for a thioacylenzyme intermediate. Control experiments assessed that reduction of the coenzyme has occurred within the crystal. The structure reveals that reduction of the cofactor upon acylation leads to an extensive motion of the nicotinamide moiety with a flip of the reduced pyridinium ring away from the active site without significant changes of the protein structure. This event positions the reduced nicotinamide moiety in a pocket that likely constitutes the exit door for NADPH. Arguments are provided that the structure reported here constitutes a reasonable picture of the first thioacylenzyme intermediate characterized thus far in the ALDH family and that the position of the reduced nicotinamide moiety observed in GAPN is the one suitable for the deacylation step within all of the nonphosphorylating CoA-independent ALDH family.     

Thermodynamic analysis of the emergence of new regulatory properties in a phosphoribulokinase-glyceraldehyde 3-phosphate dehydrogenase complex

Glyceraldehyde 3-phosphate dehydrogenase and phosphoribulokinase exist as stable enzymes and as part of a complex in Chlamydomonas reinhardtii. We show here that phosphoribulokinase exerts an imprinting on glyceraldehyde 3-phosphate dehydrogenase, which affects its catalysis by decreasing the energy barrier of the reactions with NADH or NADPH by 3.8 +/- 0.5 and 1.3 +/- 0.3 kJ.mol(-1). Phosphoribulokinase and glyceraldehyde 3-phosphate dehydrogenase within the complex are regulated by NADP(H) but not by NAD(H). The activities of the metastable phosphoribulokinase and glyceraldehyde 3-phosphate dehydrogenase released from the complex preincubated with NADP(H) are different from those of the metastable enzymes released from the untreated complex. NADP(H) increases phosphoribulokinase and NADPH-glyceraldehyde 3-phosphate dehydrogenase activities with a (~)K(0.5 (NADP)) of 0.68 +/- 0.16 mm and a (~)K(0.5 (NADPH)) of 2.93 +/- 0.87 mm and decreases NADH-dependent activity. 1 mm NADP increases the energy barrier of the NADH-glyceraldehyde 3-phosphate dehydrogenase-dependent reaction by 1.8 +/- 0.2 kJ.mol(-1) and decreases that of the reactions catalyzed by phosphoribulokinase and NADPH-glyceraldehyde 3-phosphate dehydrogenase by 3 +/- 0.2 and 1.2 +/- 0.3 kJ.mol(-1), respectively. These cofactors have no effect on the independent stable enzymes. Therefore, protein-protein interactions may give rise to new regulatory properties.     

Modification of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides with the 2',3'-dialdehyde derivative of NADP+ (oNADP+)

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides is irreversibly inactivated by the 2,3'-dialdehyde of NADP+ (oNADP+) in the absence of substrate. The inactivation is first order with respect to NADP+ concentration and follows saturation kinetics, indicating that the enzyme initially forms a reversible complex with the inhibitor followed by covalent modification (KI = 1.8 mM). NADP+ and NAD+ protect the enzyme from inactivation by oNADP+. The pK of inactivation is 8.1. oNADP+ is an effective coenzyme in assays of glucose-6-phosphate dehydrogenase (Km = 200 microM). Kinetic evidence and binding studies with [14C] oNADP+ indicate that one molecule of oNADP+ binds per subunit of glucose-6-phosphate dehydrogenase when the enzyme is completely inactivated. The interaction between oNADP+ and the enzyme does not generate a Schiff's base, or a conjugated Schiff's base, but the data are consistent with the formation of a dihydroxymorpholino derivative.     

In silico aided metabolic engineering of Saccharomyces cerevisiae for improved bioethanol production

In silico genome-scale cell models are promising tools for accelerating the design of cells with improved and desired properties. We demonstrated this by using a genome-scale reconstructed metabolic network of Saccharomyces cerevisiae to score a number of strategies for metabolic engineering of the redox metabolism that will lead to decreased glycerol and increased ethanol yields on glucose under anaerobic conditions. The best-scored strategies were predicted to completely eliminate formation of glycerol and increase ethanol yield with 10%. We successfully pursued one of the best strategies by expressing a non-phosphorylating, NADP(+)-dependent glyceraldehyde-3-phosphate dehydrogenase in S. cerevisiae. The resulting strain had a 40% lower glycerol yield on glucose while the ethanol yield increased with 3% without affecting the maximum specific growth rate. Similarly, expression of GAPN in a strain harbouring xylose reductase and xylitol dehydrogenase led to an improvement in ethanol yield by up to 25% on xylose/glucose mixtures.