Role of the histidine 176 residue in glyceraldehyde-3-phosphate dehydrogenase as probed by site-directed mutagenesis

Soukri, Abdelaziz; Mougin, Annie; Corbier, Catherine; Wonacott, A.; Branlant, Christiane; Branlant, Guy

doi:10.1021/bi00432a036

Cited by 85 publications

(67 citation statements)

References 24 publications

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“…To our knowledge, an esterase activity has been described so far only for the classical glycolytic NAD-dependent GAPDH (44). We found that Synechocystis sp.…”

mentioning

confidence: 68%

“…One unit of enzyme was defined as the amount which catalyzes the generation, or disappearance, of 1 mol of NAD(P)H per min. The esterase activity of GAPDH was determined spectrophotometrically at 30°C by monitoring the p-nitrophenol generated in the hydrolysis of p-nitrophenylesters at 400 nm (44). All kinetic parameters given represent average values of at least four independent determinations.…”

Section: Methodsmentioning

confidence: 99%

“…Due to their key metabolic roles, these proteins and their corresponding gap genes have been well characterized in many organisms (8,16). In the case of the widespread glycolytic GAPDH, by far the best-studied member of this enzyme family, the knowledge of its structure has allowed directed mutagenesis of essential amino acid residues of the active site and the eventual detailed description of the enzymatic mechanism (12,18,44).Three distinct GAPDH enzymes with different subcellular localizations and performing diverse roles are present in photosynthetic eukaryotic organisms: (i) a typical NAD-dependent glycolytic enzyme (EC 1.2.1.12), similar to that found in all organisms so far studied and located in the cytoplasm; (ii) the NADP-dependent GAPDH (EC 1.2.1.13), a key component of the reductive pentose phosphate (RPP) cycle, which is located in the chloroplast stroma (7, 42); and (iii) a cytosolic nonphosphorylating NADP-dependent GAPDH (EC 1.2.1.9), characteristic of photosynthetic eukaryotes, that has been proposed to metabolize trioses exported from the chloroplast but whose precise function remains to be established (29,41). In contrast, a single GAPDH enzyme exhibiting comparable activity levels with NAD or NADP as the cofactor (EC 1.2.1.-) has been found in photoautotrophic cyanobacteria and claimed, on the basis of preliminary biochemical data, to be involved in both photosynthetic (anabolic) and glycolytic (catabolic) carbon metabolism (23).…”

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confidence: 99%

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confidence: 99%

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Functional complementation of an Escherichia coli gap mutant supports an amphibolic role for NAD(P)-dependent glyceraldehyde-3-phosphate dehydrogenase of Synechocystis sp. strain PCC 6803

1997

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The gap-2 gene, encoding the NAD(P)-dependent D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH2) of the cyanobacterium Synechocystis sp. strain PCC 6803, was cloned by functional complementation of an Escherichia coli gap mutant with a genomic DNA library; this is the first time that this cloning strategy has been used for a GAPDH involved in photosynthetic carbon assimilation. The Synechocystis DNA region able to complement the E. coli gap mutant was narrowed down to 3 kb and fully sequenced. A single complete open reading frame of 1,011 bp encoding a protein of 337 amino acids was found and identified as the putative gap-2 gene identified in the complete genome sequence of this organism. Determination of the transcriptional start point, identification of putative promoter and terminator sites, and orientation of the truncated flanking genes suggested the gap-2 transcript should be monocystronic, a possibility further confirmed by Northern blot studies. Both natural and recombinant homotetrameric GAPDH2s were purified and found to exhibit virtually identical physicochemical and kinetic properties. The recombinant GAPDH2 showed the dual pyridine nucleotide specificity characteristic of the native cyanobacterial enzyme, and similar ratios of NAD-to NADPdependent activities were found in cell extracts from Synechocystis as well as in those from the complemented E. coli clones. The deduced amino acid sequence of Synechocystis GAPDH2 presented a high degree of identity with sequences of the chloroplastic NADP-dependent enzymes. In agreement with this result, immunoblot analysis using monospecific antibodies raised against GAPDH2 showed the presence of the 38-kDa GAPDH subunit not only in crude extracts from the gap-2-expressing E. coli clones and all cyanobacteria that were tested but also in those from eukaryotic microalgae and plants. Western and Northern blot experiments showed that gap-2 is conspicuously expressed, although at different levels, in Synechocystis cells grown in different metabolic regimens, even under chemoheterotrophic conditions. A possible amphibolic role of the cyanobacterial GAPDH2, namely, anabolic for photosynthetic carbon assimilation and catabolic for carbohydrate degradative pathways, is discussed.Phosphorylating D-glyceraldehyde-3-phosphate dehydrogenases (GAPDHs) are enzymes involved in the central pathways of carbon metabolism that have been found in all organisms studied so far (16). Due to their key metabolic roles, these proteins and their corresponding gap genes have been well characterized in many organisms (8,16). In the case of the widespread glycolytic GAPDH, by far the best-studied member of this enzyme family, the knowledge of its structure has allowed directed mutagenesis of essential amino acid residues of the active site and the eventual detailed description of the enzymatic mechanism (12,18,44).Three distinct GAPDH enzymes with different subcellular localizations and performing diverse roles are present in photosynthetic eukaryotic organisms: (i) a typical NAD-dependent...

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“…To our knowledge, an esterase activity has been described so far only for the classical glycolytic NAD-dependent GAPDH (44). We found that Synechocystis sp.…”

mentioning

confidence: 68%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Functional complementation of an Escherichia coli gap mutant supports an amphibolic role for NAD(P)-dependent glyceraldehyde-3-phosphate dehydrogenase of Synechocystis sp. strain PCC 6803

1997

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“…Knowledge of the three-dimensional structure will allow us to study, using site-directed mutagenesis, structure-function relationships. A series of mutants affecting amino acids directly related to catalysis or involved in substrate or coenzyme binding have been constructed and characterized (Soukri et al, 1989;Corbier et al, 1990;Corbier, Della Seta & Branlant, 1992, Corbier, Michels, Wonacott & Branlant, 1994. Futhermore, a comparison of the E. coli GAPDH structure with the well refined structure of the B. stearothermophilus GAPDH will help to delineate more precisely the conserved and variable parts of the structure.…”

Section: Introductionmentioning

confidence: 99%

Crystallization and preliminary X-ray diffraction studies ofEscherichia coliglyceraldehyde-3-phosphate dehydrogenase

Olivier

Buisson²,

Fanchon³

et al. 1995

Acta Cryst D

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Phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in glycolysis. Single crystals of NAD-dependent GAPDH from Escherichia coli have been obtained by vapour diffusion at room temperature using trisodium citrate as precipitant. In almost the same crystallization conditions, two kinds of crystals were found to be suitable for X-ray diffraction. The crystals with only one half of a tetramer in the asymmetric unit were chosen for highresolution analysis. They belonged to space group C2221, with cell dimensions a = 79.1, b = 189.6 and c = 122.2 A. These crystals diffracted to 1.8 A resolution.

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Tunable Bromomagnesium Thiolate Tishchenko Reaction Catalysts: Intermolecular Aldehyde–Trifluoromethylketone Coupling

Cronin¹,

Manoni²,

Connor³

et al. 2010

Angewandte Chemie

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The Tishchenko reaction [1] (discovered by Claisen [2] in 1887) is the disproportionation of two aldehyde molecules to furnish an ester product (Scheme 1). [3] Aluminum alkoxides [1,4] and boric acid, [5] were the first classes of synthetically relevant homogeneous catalysts [6] for this reaction, these were then followed by a range of transition-metal complexes of low to high catalytic activity but often limited practical utility. [7,8] More recently, lanthanide, [9] actinide, [10] and calcium [11] complexes capable of promoting aldehyde dimerization with excellent activity have been reported. A generalized mechanistic outline of the process is given in Scheme 1: reaction of the transition-metal complex 1 with the aldehyde generates the metal alkoxide 2, which acts as the hydride-transfer agent in a metal-mediated redox process (i.e. 3) leading to ester 4. [12] The Tishchenko reaction is an unusual process from a mechanistic standpoint with the potential to allow chemists to plan the synthesis of ester products through an unconventional disconnection. While recent advances in catalyst development have resulted in increased promise as a general synthetic methodology, the utility of the Tishchenko reaction is somewhat limited by two factors: a) Often the reported catalyst systems result in lower yields of isolated products from substituted benzaldehydes, and b) intermolecular crossed-Tishchenko reactions between equimolar amounts of two different carbonyl moieties are generally not possible. In particular no examples of intermolecular cross-coupling [13,14] between an aldehyde and a ketone are known, meaning that the intermolecular reaction cannot currently be utilized to generate new stereogenic centers.We were therefore encouraged to attempt the development of an alternative catalyst system for the intermolecular Tishchenko process. Our objective was to devise a simple, inexpensive, and easy to use small-molecule promoter, the steric and electronic characteristics of which could be readily tuned, with the eventual goal of broadening the scope of the Tishchenko reaction to include ketone substrates. We were inspired by the mode of action of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (G3PDHase), which promotes aldehyde oxidation through base-catalyzed addition of a cysteine residue to the aldehyde substrate to give the corresponding hemithioacetal conjugate base 5 (Scheme 2, A), which participates in an intermolecular hydride-transfer reaction with enzyme-bound NAD + . The resulting electrophilic thioester 6 then undergoes either hydrolysis or substitution by inorganic phosphate (depending on the enzyme variant). [15][16][17] We postulated the viability of an artificial process in which an analogous hemithioacetal anion 9 generated from benzaldehyde (8) and a bromomagnesium thiolate [18] could transfer hydride [19,20] to another carbonyl moiety to give magnesium alkoxide 10 and thioester 11, [21] which would subsequently couple to form the ester product 12 with regeneration of the thiolate cata...

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Role of the histidine 176 residue in glyceraldehyde-3-phosphate dehydrogenase as probed by site-directed mutagenesis

Cited by 85 publications

References 24 publications

Functional complementation of an Escherichia coli gap mutant supports an amphibolic role for NAD(P)-dependent glyceraldehyde-3-phosphate dehydrogenase of Synechocystis sp. strain PCC 6803

Functional complementation of an Escherichia coli gap mutant supports an amphibolic role for NAD(P)-dependent glyceraldehyde-3-phosphate dehydrogenase of Synechocystis sp. strain PCC 6803

Crystallization and preliminary X-ray diffraction studies ofEscherichia coliglyceraldehyde-3-phosphate dehydrogenase

Tunable Bromomagnesium Thiolate Tishchenko Reaction Catalysts: Intermolecular Aldehyde–Trifluoromethylketone Coupling

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