Engineering the allosteric properties of archaeal non-phosphorylating glyceraldehyde-3-phosphate dehydrogenases

The archaeal non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN, EC 1.2.1.9) is a highly allosteric enzyme activated by glucose 1-phosphate (Glc1P). Recent kinetic analyses of two GAPN homologs from Sulfolobales show different allosteric behaviors toward the substrate glyceraldehyde-3-phosphate (GAP) and the allosteric effector Glc1P. In GAPN from Sulfolobus tokodaii (Sto-GAPN), Glc1P-induced activation follows an increase in affinity for GAP rather than an increase in maximum velocity, whereas in GAPN from Sulfolobus solfataricus (Sso-GAPN), Glc1P-induced activation follows an increase in maximum velocity rather than in affinity for GAP. To explore the molecular basis of this difference between Sto-GAPN and Sso-GAPN, we generated 14 mutants and 2 chimeras. The analyses of chimeric GAPNs generated from regions of Sto-GAPN and Sso-GAPN indicated that a 57-residue module located in the subunit interface was clearly involved in their allosteric behavior. Among the point mutations in this modular region, the Y139R variant of Sto-GAPN no longer displayed a sigmoidal K-type-like allostery, but instead had apparent V-type allostery similar to that of Sso-GAPN, suggesting that the residue located in the center of the homotetramer critically contributes to the allosteric behavior.     

The non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) of <Emphasis Type="Italic">Sulfolobus solfataricus</Emphasis>: a key-enzyme of the semi-phosphorylative branch of the Entner–Doudoroff pathway

Archaea utilize a branched modification of the classical Entner–Doudoroff (ED) pathway for sugar degradation. The semi-phosphorylative branch merges at the level of glyceraldehyde 3-phosphate (GAP) with the lower common shunt of the Emden-Meyerhof-Parnas pathway. In Sulfolobus solfataricus two different GAP converting enzymes—classical phosphorylating GAP dehydrogenase (GAPDH) and the non-phosphorylating GAPDH (GAPN)—were identified. In Sulfolobales the GAPN encoding gene is found adjacent to the ED gene cluster suggesting a function in the regulation of the semi-phosphorylative ED branch. The biochemical characterization of the recombinant GAPN of S. solfataricus revealed that—like the well-characterized GAPN from Thermoproteus tenax—the enzyme of S. solfataricus exhibits allosteric properties. However, both enzymes show some unexpected differences in co-substrate specificity as well as regulatory fine-tuning, which seem to reflect an adaptation to the different lifestyles of both organisms. Phylogenetic analyses and database searches in Archaea indicated a preferred distribution of GAPN (and/or GAP oxidoreductase) in hyperthermophilic Archaea supporting the previously suggested role of GAPN in metabolic thermoadaptation. This work suggests an important role of GAPN in the regulation of carbon degradation via modifications of the EMP and the branched ED pathway in hyperthermophilic Archaea. Thijs J. G. Ettema and Hatim Ahmed have contributed equally to this work.     

Properties and Role of Glyceraldehyde-3-Phosphate Dehydrogenase in the Control of Fermentation Pattern and Growth in a Ruminal Bacterium, <Emphasis Type="Italic">Streptococcus bovis</Emphasis>

To clarify the control of glycolysis and the fermentation pattern in Streptococcus bovis, the molecular and enzymatic properties of NAD⁺-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were examined. The GAPDH gene (gapA) was found to cluster with several others, including those that encode phosphoglycerate kinase and translation elongation factor G, however, gapA was transcribed in a monocistronic fashion. Since biochemical properties, such as optimal pH and affinity for glyceraldehyde-3-phosphate (GAP), were not very different between GAPDH- and NADP⁺-specific glyceraldehyde-3-phosphate dehydrogenase (GAPN), the flux from GAP may be greatly influenced by the relative amounts of these two enzymes. Using S. bovis JB1 as a parent, JB1gapA and JB1ldh, which overproduce GAPDH and lactate dehydrogenase (LDH), respectively, were constructed to examine the control of the glycolytic flux and lactate production. There were no significant differences in growth rates and formate-to-lactate ratios among JB1, JB1gapA, and JB1ldh grown on glucose. When grown on lactose, JB1ldh showed a much lower formate-to-lactate ratio than JB1gapA, which showed the highest NADH-to-NAD⁺ ratio. However, growth rates did not differ among JB1, JB1gapA, and JB1ldh. These results suggest that GAPDH is not involved in the control of the glycolytic flux and that lactate production is mainly controlled by LDH activity.     

Unraveling the function of paralogs of the aldehyde dehydrogenase super family from Sulfolobus solfataricus

Aldehyde dehydrogenases (ALDHs) have been well established in all three domains of life and were shown to play essential roles, e.g., in intermediary metabolism and detoxification. In the genome of Sulfolobus solfataricus, five paralogs of the aldehyde dehydrogenases superfamily were identified, however, so far only the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) and α-ketoglutaric semialdehyde dehydrogenase (α-KGSADH) have been characterized. Detailed biochemical analyses of the remaining three ALDHs revealed the presence of two succinic semialdehyde dehydrogenase (SSADH) isoenzymes catalyzing the NAD(P)⁺-dependent oxidation of succinic semialdehyde. Whereas SSO1629 (SSADH-I) is specific for NAD⁺, SSO1842 (SSADH-II) exhibits dual cosubstrate specificity (NAD(P)⁺). Physiological significant activity for both SSO-SSADHs was only detected with succinic semialdehyde and α-ketoglutarate semialdehyde. Bioinformatic reconstructions suggest a major function of both enzymes in γ-aminobutyrate, polyamine as well as nitrogen metabolism and they might additionally also function in pentose metabolism. Phylogenetic studies indicated a close relationship of SSO-SSALDHs to GAPNs and also a convergent evolution with the SSADHs from E. coli. Furthermore, for SSO1218, methylmalonate semialdehyde dehydrogenase (MSDH) activity was demonstrated. The enzyme catalyzes the NAD⁺- and CoA-dependent oxidation of methylmalonate semialdehyde, malonate semialdehyde as well as propionaldehyde (PA). For MSDH, a major function in the degradation of branched chain amino acids is proposed which is supported by the high sequence homology with characterized MSDHs from bacteria. This is the first report of MSDH as well as SSADH isoenzymes in Archaea.     

The crystal structure of galactitol-1-phosphate 5-dehydrogenase from Escherichia coli K12 provides insights into its anomalous behavior on IMAC processes

Endogenous galactitol-1-phosphate 5-dehydrogenase (GPDH) (EC 1.1.1.251) from Escherichia coli spontaneously interacts with Ni²⁺-NTA matrices becoming a potential contaminant for recombinant, target His-tagged proteins. Purified recombinant, untagged GPDH (rGPDH) converted galactitol into tagatose, and d-tagatose-6-phosphate into galactitol-1-phosphate, in a Zn²⁺- and NAD(H)-dependent manner and readily crystallized what has permitted to solve its crystal structure. In contrast, N-terminally His-tagged GPDH was marginally stable and readily aggregated. The structure of rGPDH revealed metal-binding sites characteristic from the medium-chain dehydrogenase/reductase protein superfamily which may explain its ability to interact with immobilized metals. The structure also provides clues on the harmful effects of the N-terminal His-tag.

Structured summary of protein interactions

GPDH and GPDHbind by molecular sieving (View interaction)GPDH and GPDHbind by x-ray crystallography (View interaction)GPDH and GPDHbind by cosedimentation in solution (View interaction)     

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.     

Archaeal Aldehyde Dehydrogenase ST0064 from Sulfolobus tokodaii,a Paralog of Non-Phosphorylating Glyceraldehyde-3-phosphate Dehydrogenase,Is a Succinate Semialdehyde Dehydrogenase

Aldehyde dehydrogenase ST0064, the closest paralog of previously characterized allosteric non-phosphorylating glyceraldehyde-3-phosphate (GAP) dehydrogenase (GAPN, ST2477) from a thermoacidophilic archaeon, Sulfolobus tokodaii, was expressed heterologously and characterized in detail. ST0064 showed remarkable activity toward succinate semialdehyde (SSA) (K _m of 0.0029 mM and k _cat of 30.0 s^?1) with no allosteric regulation. Activity toward GAP was lower (K _m of 4.6 mM and k _cat of 4.77 s^?1), and previously predicted succinyl-CoA reductase activity was not detected, suggesting that the enzyme functions practically as succinate semialdehyde dehydrogenase (SSADH). Phylogenetic analysis indicated that archaeal SSADHs and GAPNs are closely related within the aldehyde dehydrogenase superfamily, suggesting that they are of the same origin.     

Characterization of the glyceraldehyde 3-phosphate dehydrogenase from the extremely halophilic archaebacterium Haloarcula vallismortis

Glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) from the extremely halophilic archaebacterium Haloarcula vallismortis has been purified in a four step procedure to electrophoretic homogeneity. The enzyme is a tetramer with a relative molecular mass of 160000. It is strictly NAD⁺-dependent and exhibits its highest activity in 2 mol/l KCl at 45°C. Amino acid analysis and isoelectric focusing indicate an excess of acidic amino acids. Two parts of the primary sequence are reported. These peptides have been compared with glyceraldehyde 3-phosphate dehydrogenases from other archaebacteria, eubacteria and eucaryotes. The peptides show a high grade of similarity to glyceraldehyde 3-phosphate dehydrogenase from eucaryotes.Abbreviations BCA bicinchoninic acid - CTAB cetyltrimethyl ammonium bromide - DTE dithioerythritol - DTT dithiothreitol - GAP glyccraldehyde 3-phosphate - GAPDH glyceraldehyde 3-phosphate dehydrogenase     

Glyceraldehyde-3-phosphate dehydrogenase from human erythrocyte membranes. Kinetic mechanism and competitive substrate inhibition by glyceraldehyde 3-phosphate

Glyceraldehyde-3-phosphate dehydrogenase (GAPD) was isolated from human erythrocyte ghosts by a simple procedure utilizing ammonium sulfate precipitation and affinity chromatography on NAD⁺-Sepharose 4B. The purified enzyme had a specific activity of 98 units/mg protein. The kinetic mechanism of GAPD was studied by product and deadend inhibition using NADH, α-glycerophosphate, nitrate, and 2,3-diphosphoglycerate. The results indicated that the human erythrocyte GAPD-catalyzed reaction follows an ordered ter bi mechanism characterized by the sequential addition of NAD⁺, glyceraldehyde 3-phosphate (GAP), and phosphate to the enzyme and the sequential release of 1,3-diphosphoglycerate and NADH from the enzyme. This contrasts with the mechanism (rapid equilibrium random ter bi) proposed by Oguchi (1970, J. Biochem. (Tokyo)68, 427–439) who based his conclusion on the initial rate data alone. Since the Michaelis-Menten kinetics were not applicable to this enzyme because of the competitive substrate inhibition by GAP, we devised a new kinetic approach for determining the parameters of the GAPD-catalyzed reaction. Results of this study indicate that the GAPD-catalyzed reaction is regulated by both ATP and GAP. We propose that GAP acts as an “amplifier” for the feedback inhibition effect of ATP. We discuss the effect this may have played in causing controversy over the regulatory role of this enzyme in glycolysis.     

Improved Production of 2,3-Butanediol in Bacillus amyloliquefaciens by Over-Expression of Glyceraldehyde-3-Phosphate Dehydrogenase and 2,3-butanediol Dehydrogenase

Background

Previously, a safe strain, Bacillus amyloliquefaciens B10-127 was identified as an excellent candidate for industrial-scale microbial fermentation of 2,3-butanediol (2,3-BD). However, B. amyloliquefaciens fermentation yields large quantities of acetoin, lactate and succinate as by-products, and the 2,3-BD yield remains prohibitively low for commercial production.

Methodology/Principal Findings

In the 2,3-butanediol metabolic pathway, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the conversion of 3-phosphate glyceraldehyde to 1,3-bisphosphoglycerate, with concomitant reduction of NAD⁺ to NADH. In the same pathway, 2,3-BD dehydrogenase (BDH) catalyzes the conversion of acetoin to 2,3-BD with concomitant oxidation of NADH to NAD⁺. In this study, to improve 2,3-BD production, we first over-produced NAD⁺-dependent GAPDH and NADH-dependent BDH in B. amyloliquefaciens. Excess GAPDH reduced the fermentation time, increased the 2,3-BD yield by 12.7%, and decreased the acetoin titer by 44.3%. However, the process also enhanced lactate and succinate production. Excess BDH increased the 2,3-BD yield by 16.6% while decreasing acetoin, lactate and succinate production, but prolonged the fermentation time. When BDH and GAPDH were co-overproduced in B. amyloliquefaciens, the fermentation time was reduced. Furthermore, in the NADH-dependent pathways, the molar yield of 2,3-BD was increased by 22.7%, while those of acetoin, lactate and succinate were reduced by 80.8%, 33.3% and 39.5%, relative to the parent strain. In fed-batch fermentations, the 2,3-BD concentration was maximized at 132.9 g/l after 45 h, with a productivity of 2.95 g/l·h.

Conclusions/Significance

Co-overexpression of bdh and gapA genes proved an effective method for enhancing 2,3-BD production and inhibiting the accumulation of unwanted by-products (acetoin, lactate and succinate). To our knowledge, we have attained the highest 2,3-BD fermentation yield thus far reported for safe microorganisms.     

Trypanosoma evansi is alike to Trypanosoma brucei brucei in the subcellular localisation of glycolytic enzymes

Trypanosoma evansi, which causes surra, is descended from Trypanosoma brucei brucei, which causes nagana. Although both parasites are presumed to be metabolically similar, insufficient knowledge of T. evansi precludes a full comparison. Herein, we provide the first report on the subcellular localisation of the glycolytic enzymes in T. evansi, which is a alike to that of the bloodstream form (BSF) of T. b. brucei: (i) fructose-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hexokinase, phosphofructokinase, glucose-6-phosphate isomerase, phosphoglycerate kinase, triosephosphate isomerase (glycolytic enzymes) and glycerol-3-phosphate dehydrogenase (a glycolysis-auxiliary enzyme) in glycosomes, (ii) enolase, phosphoglycerate mutase, pyruvate kinase (glycolytic enzymes) and a GAPDH isoenzyme in the cytosol, (iii) malate dehydrogenase in cytosol and (iv) glucose-6-phosphate dehydrogenase in both glycosomes and the cytosol. Specific enzymatic activities also suggest that T. evansi is alike to the BSF of T. b. brucei in glycolytic flux, which is much faster than the pentose phosphate pathway flux, and in the involvement of cytosolic GAPDH in the NAD⁺/NADH balance. These similarities were expected based on the close phylogenetic relationship of both parasites.     

Glyceraldehyde-3-phosphate dehydrogenase (NADP) from Sinapis alba: Steady state kinetics

Substrate interaction and product inhibition kinetics of the forward reaction of glyceraldehyde-3-phosphate dehydrogenase (NADP) (EC 1.2.1.13) from Sinapis alba suggest an Uni Uni Uni Bi Ping Pong mechanism (NAD(P)H on, glyceraldehyde-3-phosphate off, 1,3-diphosphoglycerate on, phosphate off, NAD(P)⁺ off) with an apparent Theorell Chance displacement between 1,3-diphosphoglycerate and phosphate. The proposed mechanism predicts the existence of stable enzyme-NAD(P)⁺ and acyl-enzyme complexes as obligatory intermediates. A comparison of the present findings on the NADP-enzyme with an earlier kinetic analysis of the NAD-specific enzyme from plants (EC 1.2.1.12) by other authors shows that the kinetic mechanisms for the two enzymes, although similar in principle (both show Ping Pong kinetics), differ in some details.     

Oxidation of C1-compounds in Pseudomonas C

Extracts of Pseudomonas C grown on methanol as sole carbon and energy source contain a methanol dehydrogenase activity which can be coupled to phenazine methosulfate. This enzyme catalyzes two reactions namely the conversion of methanol to formaldehyde (phenazine methosulfate coupled) and the oxidation of formaldehyde to formate (2,6-dichloroindophenol-coupled). Activities of glutathione-dependent formaldehyde dehydrogenase (NAD⁺) and formate dehydrogenase (NAD⁺) were also detected in the extracts.The addition of d-ribulose 5-phosphate to the reaction mixtures caused a marked increase in the formaldehyde-dependent reduction of NAD⁺ or NADP⁺. In addition, the oxidation of [¹⁴C]formaldehyde to CO₂, by extracts of Pseudomonas C, increased when d-ribulose 5-phosphate was present in the assay mixtures.The amount of radioactivity found in CO₂, was 6.8-times higher when extracts of methanol-grown Pseudomona C were incubated for a short period of time with [1-¹⁴C]glucose 6-phosphate than with [U-¹⁴C]glucose 6-phosphate.These data, and the presence of high specific activities of hexulose phosphate synthase, phosphoglucoisomerase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase indicate that in methanol-grown Pseudomonas C, formaldehyde carbon is oxidized to CO₂ both via a cyclic pathway which includes the enzymes mentioned and via formate as an oxidation intermediate, with the former predominant.     

Dimerization, but not phosphothreonine binding, is conserved between the forkhead-associated domains of Drosophila MU2 and human MDC1

Mutator 2 (MU2) in Drosophila melanogaster has been proposed to be the ortholog of human MDC1, a key mediator in DNA damage response. The forkhead-associated (FHA) domain of MDC1 is a dimerization module regulated by trans binding to phosphothreonine 4 from another molecule. Here we present the crystal structure of the MU2 FHA domain at 1.9 Å resolution, revealing its evolutionarily conserved role in dimerization. As compared to the MDC1 FHA domain, the MU2 FHA domain dimerizes using a different and more stable interface and contains a degenerate phosphothreonine-binding pocket. Our results suggest that the MU2 dimerization is constitutive and lacks phosphorylation-mediated regulation.Structured summary of protein interactionsMU2 and MU2 bind by cosedimentation in solution (View interaction)MU2 and MU2 bind by X-ray crystallography (View interaction)MU2 and MU2 bind by molecular sieving (View interaction)     

Study of the Properties of Phosphorylating D-Glyceraldehyde-3-phosphate Dehydrogenase

The properties of the active center of phosphorylating D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are considered with emphasis on the structure of anion-binding sites and their role in catalysis. The results of studies on the molecular mechanism of the effect of NAD⁺ on the enzyme conformation are discussed. Experimental evidence is presented supporting the idea that negative cooperativity of NAD⁺ binding and half-of-the-sites reactivity exhibited by GAPDH are generated by different mechanisms. Data obtained with rabbit muscle and Escherichia coli GAPDH point to preexisting asymmetry in these tetramers. Structural determinants that can control the transition of the tetramer from the symmetric to the asymmetric state were found.     

Mechanistic characterization of sulfur transfer from cysteine desulfurase SufS to the iron-sulfur scaffold SufU in Bacillus subtilis

Iron–sulfur cluster biosynthesis in Gram-positive bacteria is mediated by the SUF system. The transfer of sulfide from the cysteine desulfurase SufS to the scaffold protein SufU is one of the first steps within the assembly process. In this study, we analyzed the interaction between Bacillus subtilis SufS and its scaffold SufU. The activity of SufS represents a Ping-Pong mechanism leading to successive sulfur loading of the conserved cysteine residues in SufU. Cysteine 41 of SufU is shown to be essential for receiving sulfide from SufS, while cysteines 66 and 128 are needed for SufS/SufU interaction. In conclusion, we present the first step-by-step model for loading of the essential scaffold component SufU by its sulfur donor SufS.

Structured summary

SufS and SufUbind by molecular sieving(View interaction)SufSbinds to SufS by molecular sieving(View interaction)SufS and SufUredox react by enzymatic study (View Interaction 1, 2, 3, 4, 5)SufUphysically interacts with SufS by pull down (View Interaction 1, 2)     

Brownian dynamics simulations of aldolase binding glyceraldehyde 3-phosphate dehydrogenase and the possibility of substrate channeling

Brownian dynamics (BD) simulations test for channeling of the substrate, glyceraldehyde 3-phosphate (GAP), as it passes between the enzymes fructose-1,6-bisphosphate aldolase (aldolase) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). First, BD simulations determined the favorable complexes between aldolase and GAPDH; two adjacent subunits of GAPDH form salt bridges with two subunits of aldolase. These intermolecular contacts provide a strong electrostatic interaction between the enzymes. Second, BD simulates GAP moving out of the active site of the A or D aldolase subunit and entering any of the four active sites of GAPDH. The efficiency of transfer is determined as the relative number of BD trajectories that reached any active site of GAPDH. The distribution functions of the transfer time were calculated based on the duration of successful trajectories. BD simulations of the GAP binding from solution to aldolase/GAPDH complex were compared to the channeling simulations. The efficiency of transfer of GAP within an aldolase/GAPDH complex was 2 to 3% compared to 1.3% when GAP was binding to GAPDH from solution. There is a preference for GAP channeling between aldolase and GAPDH when compared to binding from solution. However, this preference is not large enough to be considered as a theoretical proof of channeling between these proteins.     

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.