Crystallization and preliminary X-ray study of chloroplast glyceraldehyde-3-phosphate dehydrogenase

Sabatino, Piera; Fermani, Simona; Ripamonti, A.; Cassetta, Alberto; Scagliarini, Sandra; Trost, Paolo

doi:10.1107/s090744499801302x

Cited by 6 publications

(3 citation statements)

References 19 publications

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“…In any event, very large amounts of A 4 -GAPDH were obtained from spinach chloroplasts to grow crystals for structural analysis (Sabatino et al 1999) and the tridimensional structure of native A 4 -GAPDH from spinach is still the only one available among photosynthetic GAPDHs (Fermani et al 2001). The overall structure ( Fig.…”

Section: Diversity and Evolution Of Gapdh Subunits And Isozymesmentioning

confidence: 99%

Thioredoxin-dependent regulation of photosynthetic glyceraldehyde-3-phosphate dehydrogenase: autonomous vs. CP12-dependent mechanisms

et al. 2006

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Regulation of the Calvin-Benson cycle under varying light/dark conditions is a common property of oxygenic photosynthetic organisms and photosynthetic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is one of the targets of this complex regulatory system. In cyanobacteria and most algae, photosynthetic GAPDH is a homotetramer of GapA subunits which do not contain regulatory domains. In these organisms, dark-inhibition of the Calvin-Benson cycle involves the formation of a kinetically inhibited supramolecular complex between GAPDH, the regulatory peptide CP12 and phosphoribulokinase. Conditions prevailing in the dark, i.e. oxidation of thioredoxins and low NADP(H)/NAD(H) ratio promote aggregation. Although this regulatory system has been inherited in higher plants, these phototrophs contain in addition a second type of GAPDH subunits (GapB) resulting from the fusion of GapA with the C-terminal half of CP12. Heterotetrameric A(2)B(2)-GAPDH constitutes the major photosynthetic GAPDH isoform of higher plants chloroplasts and coexists with CP12 and A(4)-GAPDH. GapB subunits of A(2)B(2)-GAPDH have inherited from CP12 a regulatory domain (CTE for C-terminal extension) which makes the enzyme sensitive to thioredoxins and pyridine nucleotides, resembling the GAPDH/CP12/PRK system. The two systems are similar in other respects: oxidizing conditions and low NADP(H)/NAD(H) ratios promote aggregation of A(2)B(2)-GAPDH into strongly inactivated A(8)B(8)-GAPDH hexadecamers, and both CP12 and CTE specifically affect the NADPH-dependent activity of GAPDH. The alternative, lower activity with NADH is always unaffected. Based on the crystal structure of spinach A(4)-GAPDH and the analysis of site-specific mutants, a model of the autonomous (CP12-independent) regulatory mechanism of A(2)B(2)-GAPDH is proposed. Both CP12 and CTE seem to regulate different photosynthetic GAPDH isoforms according to a common and ancient molecular mechanism.

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Section: Diversity and Evolution Of Gapdh Subunits And Isozymesmentioning

confidence: 99%

Thioredoxin-dependent regulation of photosynthetic glyceraldehyde-3-phosphate dehydrogenase: autonomous vs. CP12-dependent mechanisms

et al. 2006

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“…They were processed and scaled using DENZO and SCALEPACK programs (33). The crystals belong to the space group C222, and they are isomorphous with those of the native A 4 -GAPDH complexed with NADP (34). The unit cell parameters and the data collection statistics are shown in Table 1.…”

Section: Methodsmentioning

confidence: 99%

Dual Coenzyme Specificity of Photosynthetic Glyceraldehyde-3-phosphate Dehydrogenase Interpreted by the Crystal Structure of A₄ Isoform Complexed with NAD^,

et al. 2003

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Photosynthetic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Spinacia oleracea belongs to a wide group of GAPDHs found in most organisms displaying oxygenic photosynthesis, including cyanobacteria, green and red algae, and higher plants. As a major catalytic difference with respect to glycolytic GAPDH, photosynthetic GAPDH exhibits dual cofactor specificity toward pyridine nucleotides with a preference for NADP(H). Here we report the crystal structure of NAD-complexed recombinant A(4)-GAPDH (NAD-A(4)-GAPDH) from Spinacia oleracea, expressed in Escherichia coli. Its superimposition onto native A(4)-GAPDH complexed with NADP (NADP-A(4)-GAPDH) pinpoints specific conformational changes resulting from cofactor replacement. In photosynthetic NAD-A(4)-GAPDH, the side chain of Asp32 is oriented toward the coenzyme to interact with the adenine ribose diol, similar to glycolytic GAPDHs (NAD-specific). On the contrary, in NADP-A(4)-GAPDH Asp32 moves away to accommodate the additional 2'-phosphate group of the coenzyme and to minimize electrostatic repulsion. Asp32 rotation is allowed by the presence of the small residue Ala40, conserved in most photosynthetic GAPDHs, replacing bulky amino acid side chains in glycolytic GAPDHs. While in NADP-A(4)-GAPDH two amino acids, Thr33 and Ser188, are involved in hydrogen bonds with the 2'-phosphate group of NADP, in the NAD-complexed enzyme these interactions are lacking. The crystallographic structure of NAD-A(4)-GAPDH highlights that four residues, Thr33, Ala40, Ser188, and Ala187 (Leu, Leu, Pro, and Leu respectively, in glycolytic Bacillus stearothermophilus GAPDH sequence) are of primary importance for the dual cofactor specificity of photosynthetic GAPDH. These modifications seem to trace the minimum evolutionary route for a primitive NAD-specific GAPDH to be converted into the NADP-preferring enzyme of oxygenic photosynthetic organisms.

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“…The glycosomal GAPDHs of Trypanosoma brucei (Vellieux et al, 1993), Leishmania mexicana (Kim et al, 1995) and Tryponasoma cruzi (Souza et al, 1998) have been studied. More recently, crystallographic analyses of GAPDHs from Palinurus versicolor (Song et al, 1998), Saccharomyces cerevisiae (Gilboa et al, 1998), spinach chloroplasts (Sabatino et al, 1999;Fermani et al, 2001), archaeal GAPDH from Sulfolobus solfataricus (Fleming et al, 1998;Isupov et al, 1999) and Methanothermus fervidus (Charron et al, 1999) have been reported. For three GAPDH structures, Saccharomyces cerevisiae (Gilboa et al, 1998), Thermoproteus tenax (Brunner et al, 2000) and Synechococcus (Nakamura et al, 2001), only preliminary crystallographic analyses are available.…”

Section: Introductionmentioning

confidence: 99%

The structure of glyceraldehyde 3-phosphate dehydrogenase fromAlcaligenes xylosoxidansat 1.7 Å resolution

Antonyuk

Eady

Strange

et al. 2003

Acta Crystallogr D Biol Cryst

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The enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the Gram-negative denitrifying bacterial species Alcaligenes xylosoxidans was purified and crystallized as a contaminant protein during purification of nitrous oxide reductase. This is the first structure of a GAPDH from a denitrifying species. The crystal structure was solved at 1.7 A resolution by molecular replacement using the structure of GAPDH from Bacillus stearothermophilus as a starting model. The quality of the structure enabled the amino-acid sequence of the A. xylosoxidans GAPDH to be assigned. The structure is that of the apo-enzyme, lacking the NAD+ cofactor and with the active-site residue Cys154 oxidized. The global structure of the enzyme has a homotetrameric quaternary structure similar to that observed for its bacterial and eukaryotic counterparts. The essential role of Cys154 in the enzyme activity has been confirmed. In monomer O two half-occupancy sulfate ions were found at the active site, which are analogous to the substrate and the "attacking" phosphate seen in B. stearothermophilus. One half-occupancy sulfate ion is also located in the substrate-binding site of monomer P.

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Crystallization and preliminary X-ray study of chloroplast glyceraldehyde-3-phosphate dehydrogenase

Cited by 6 publications

References 19 publications

Thioredoxin-dependent regulation of photosynthetic glyceraldehyde-3-phosphate dehydrogenase: autonomous vs. CP12-dependent mechanisms

Thioredoxin-dependent regulation of photosynthetic glyceraldehyde-3-phosphate dehydrogenase: autonomous vs. CP12-dependent mechanisms

Dual Coenzyme Specificity of Photosynthetic Glyceraldehyde-3-phosphate Dehydrogenase Interpreted by the Crystal Structure of A₄ Isoform Complexed with NAD^,

The structure of glyceraldehyde 3-phosphate dehydrogenase fromAlcaligenes xylosoxidansat 1.7 Å resolution

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Crystallization and preliminary X-ray study of chloroplast glyceraldehyde-3-phosphate dehydrogenase

Cited by 6 publications

References 19 publications

Thioredoxin-dependent regulation of photosynthetic glyceraldehyde-3-phosphate dehydrogenase: autonomous vs. CP12-dependent mechanisms

Thioredoxin-dependent regulation of photosynthetic glyceraldehyde-3-phosphate dehydrogenase: autonomous vs. CP12-dependent mechanisms

Dual Coenzyme Specificity of Photosynthetic Glyceraldehyde-3-phosphate Dehydrogenase Interpreted by the Crystal Structure of A4 Isoform Complexed with NAD,

The structure of glyceraldehyde 3-phosphate dehydrogenase fromAlcaligenes xylosoxidansat 1.7 Å resolution

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Dual Coenzyme Specificity of Photosynthetic Glyceraldehyde-3-phosphate Dehydrogenase Interpreted by the Crystal Structure of A₄ Isoform Complexed with NAD^,