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1.
    
The NADP‐dependent glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) of Synechococcus PCC 7942 was crystallized in two different forms by the hanging‐drop vapour‐diffusion method using ammonium sulfate as a precipitant. Form I crystals were hexagonal, space group P65 or P61, with unit‐cell parameters a = b = 91.1, c = 428.6 Å, γ = 120°. Form II crystals were monoclinic, space group C2, with unit‐cell parameters a = 152.3, b = 80.9, c = 213.6 Å, β = 103.1°. Native data were collected from a frozen crystal of form I to a resolution of 2.8 Å using synchrotron radiation at SPring‐8, whereas form II crystals were easily damaged by radiation at room temperature and increased mosaicity in cryoprotectant solutions. A molecular‐replacement solution of the form I crystal was obtained in space group P65 using the program AMoRe and the structure of the NAD‐dependent GAPDH from Bacillus stearothermophilus.  相似文献   

2.
    
The classical glycolytic pathway contains an NAD‐dependent glyceraldehyde‐3‐phosphate dehydrogenase, with NADP‐dependent forms reserved for photosynthetic organisms and archaea. Here, the cloning, expression, purification, crystallization and preliminary X‐ray analysis of an NADP‐dependent glyceraldehyde‐3‐phosphate dehydrogenase from Helicobacter pylori is reported; crystals of the protein were grown both in the presence and the absence of NADP.  相似文献   

3.
    
The genome of the enteric pathogen Campylobacter jejuni encodes a single glyceraldehyde‐3‐phosphate dehydrogenase that can utilize either NADP+ or NAD+ as coenzymes for the oxidative phosphorylation of glyceraldehyde‐3‐phosphate to 1,3‐diphosphoglycerate. Here, the cloning, expression, purification, crystallization and preliminary X‐ray analysis of both the wild type and an active‐site mutant of the enzyme are presented. Preliminary X‐ray analysis revealed that in both cases the crystals diffracted to beyond 1.9 Å resolution. The space group is shown to be I4122, with unit‐cell parameters a = 90.75, b = 90.75, c = 225.48 Å, α = 90.46, β = 90.46, γ = 222.79°; each asymmetric unit contains only one subunit of the tetrameric enzyme.  相似文献   

4.
    
Glyceraldehyde‐3‐phosphate dehydrogenase from Antheraea mylitta (AmGAPDH) was cloned in pQE30 vector, overexpressed in Escherichia coli M15 (pREP4) cells and purified to homogeneity. The protein was crystallized using the hanging‐drop vapour‐diffusion method. The crystals belonged to the orthorhombic space group I222, with unit‐cell parameters a = 85.81, b = 133.72, c = 220.37 Å. X‐ray diffraction data were collected and processed to a maximum resolution of 2.2 Å. The presence of three molecules in the asymmetric unit gave a Matthews coefficient (VM) of 2.80 Å3 Da−1, with a solvent content of 56.08%.  相似文献   

5.
    
The crystal structure of the A4 isoform of photosynthetic glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) from Arabidopsis thaliana, expressed in recombinant form and complexed with NAD, is reported. The crystals, which were grown in 2.4 M ammonium sulfate and 0.1 M sodium citrate, belonged to space group I222. The asymmetric unit includes ten subunits, i.e. two independent tetramers plus a dimer that generates a third tetramer by a crystallographic symmetry operation. The crystal structure was solved by molecular replacement and refined to an R factor of 23.7% and an Rfree factor of 28.9% at 2.6 Å resolution. In the final model, each subunit binds one NAD+ molecule and two sulfates, which occupy the Ps and the Pi anion‐binding sites. Detailed knowledge of this structure is instrumental for structural investigation of supramolecular complexes of A4‐GAPDH, phosphoribulokinase and CP12, which are involved in the regulation of photosynthesis in the model plant A. thaliana.  相似文献   

6.
    
Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) is a conserved cytosolic enzyme, which plays a key role in glycolysis. GAPDH catalyzes the oxidative phosphorylation of D‐glyceraldehyde 3‐phosphate using NAD or NADP as a cofactor. In addition, GAPDH localized on the surface of some bacteria is thought to be involved in macromolecular interactions and bacterial pathogenesis. GAPDH on the surface of group B streptococcus (GBS) enhances bacterial virulence and is a potential vaccine candidate. Here, the crystal structure of GBS GAPDH from Streptococcus agalactiae in complex with NAD is reported at 2.46 Å resolution. Although the overall structure of GBS GAPDH is very similar to those of other GAPDHs, the crystal structure reveals a significant difference in the area spanning residues 294–307, which appears to be more acidic. The amino‐acid sequence of this region of GBS GAPDH is also distinct compared with other GAPDHs. This region therefore may be of interest as an immunogen for vaccine development.  相似文献   

7.
    
Helicobacter pylori is a dangerous human pathogen that resides in the upper gastrointestinal tract. Little is known about its metabolism and with the onset of antibiotic resistance new treatments are required. In this study, the expression, purification, crystallization and preliminary X‐ray diffraction of an NAD‐dependent glyceraldehyde‐3‐phosphate dehydrogenase from H. pylori are reported.  相似文献   

8.
    
The crystal structure of NADP‐dependent glyceraldehyde‐3‐phosphate dehydrogenase (NADP‐GAPDH) from Synechococcus PCC 7942 (S. 7942) in complex with NADP was solved by molecular replacement and refined to an R factor of 19.1% and a free R factor of 24.0% at 2.5 Å resolution. The overall structure of NADP‐GAPDH from S. 7942 was quite similar to those of other bacterial and eukaryotic GAPDHs. The nicotinamide ring of NADP, which is involved in the redox reaction, was oriented toward the catalytic site. The 2′‐­phosphate O atoms of NADP exhibited hydrogen bonds to the hydroxyl groups of Ser194 belonging to the S‐loop and Thr37. These residues are therefore considered to be essential in the discrimination between NADP and NAD molecules. The C‐terminal region was estimated to have an extremely flexible conformation and to play an important role in the formation of the supramolecular complex phosphoribulokinase (PRK)–regulatory peptide (CP12)–GAPDH, which regulates enzyme activities.  相似文献   

9.
    
Human liver glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) was purified and crystallized using PEG 3350 as a precipitant. However, the crystals were extremely fragile towards osmotic shock. A 1% change in PEG 3350 content causes destruction of the crystals. After many trials for freezing the crystals, X‐ray diffraction data from a native crystal were collected at 2.8 Å resolution using as a cryoprotectant a mixture consisting of paraffin oil and Paratone‐N in a 3:1 ratio and a cryoloop covered with Formver film. Crystals belong to space group P21, with unit‐cell parameters a = 63.23, b = 97.84, c = 84.23 Å, β = 104.1°. Molecular replacement with a starting model consisting of a homology model based on the low‐resolution structure of human skeletal muscle GAPDH, which has 90% identical residues with the liver protein, led to a solution. Most of the current model was assigned properly in the electron‐density map, but the map corresponding to some important regions containing the phosphate‐binding loop was ambiguous. It is planned to crystallize human liver GAPDH in the presence of phosphate ions and/or some kind of inhibitor in order to fix the flexible region.  相似文献   

10.
    
Glyceraldehyde‐3‐phosphate dehydrogenase 1 from methicillin‐resistant Staphylococcus aureus (MRSA252) was cloned in pQE30 vector, overexpressed in Escherichia coli M15(pREP4) cells and purified to homogeneity. The protein was crystallized using the hanging‐drop vapour‐diffusion method. The crystals belonged to space group P21, with unit‐cell parameters a = 65.23, b = 95.58, c = 87.91 Å, β = 106.5°. X‐ray diffraction data were collected and processed to a maximum resolution of 2.0 Å. The presence of one tetramer in the asymmetric unit gave a Matthews coefficient (VM) of 1.78 Å3 Da−1 and a solvent content of 31%. The structure was solved by molecular replacement and structure refinement is now in progress.  相似文献   

11.
    
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12.
    
Glyceraldehyde‐3‐phosphate dehydrogenase 1 (GAP1) from methicillin‐resistant Staphylococcus aureus (MRSA252) has been purified to homogeneity in the apo form. The protein was crystallized using the hanging‐drop vapour‐diffusion method. The crystals belonged to space group P21, with unit‐cell parameters a = 69.95, b = 93.68, c = 89.05 Å, β = 106.84°. X‐ray diffraction data have been collected and processed to a maximum resolution of 2.2 Å. The presence of one tetramer in the asymmetric unit gives a Matthews coefficient (VM) of 1.81 Å3 Da−1 with a solvent content of 32%. The structure has been solved by molecular replacement and structure refinement is now in progress.  相似文献   

13.
  总被引：1，自引：0，他引：1  
Thirty-seven kilodalton glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Edwardsiella tarda was suggested to be an effective vaccine candidate against E. tarda infection in previous research. For developing a vaccine, obtaining GAPDH in large quantities is necessary. In this study, we determined the complete nucleotide sequence of the gene that encodes GAPDH of E. tarda, and overexpressed the GAPDH of E. tarda by using the Escherichia coli expression system. We immunized Japanese flounder with recombinant GAPDH (rGAPDH) and evaluated its vaccine efficacy. Our results showed that rGAPDH effectively protected Japanese flounder from experimental E. tarda infection, and will contribute to the development of a vaccine against E. tarda.  相似文献   

14.
    
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 Å 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.  相似文献   

15.
    
Monoclonal antibody (Mab) 8B7 was shown in a previous study to inhibit protein translation in lysates of Sf21 cells. The antibody was thought to be specific for a 60-kDa form of elongation factor-1 alpha (EF-1alpha), primarily because the antigen immunoprecipitated by Mab 8B7 cross-reacted with Mab CBP-KK1, an antibody generated to EF-1alpha from Trypanosoma brucei. The purpose of the current study was to investigate further the antigenic specificity of Mab 8B7. The concentration of the 60-kDa antigen relative to total cellular protein proved insufficient for its definitive identification. However, subcellular fractionation of Sf21 cells yielded an additional protein of 37 kDa in the cytosolic and microsomal fractions that was reactive with Mab 8B7. The 37-kDa protein could be easily visualized by colloidal Coomassie Blue G-250 staining as a series of pI 6.9-8.4 spots on two-dimensional gels. Excision of an abundant immunoreactive spot enabled identification of the protein as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and protein database searching. Subsequent immunoblotting of purified rabbit skeletal muscle GAPDH with Mab 8B7 confirmed the antibody's specificity for GAPDH. Besides the pivotal role GAPDH plays in glycolysis, the enzyme has a number of noncanonical functions, including binding to mRNA and tRNA. The ability of Mab 8B7 to disrupt these lesser-known functions of GAPDH may account for the antibody's inhibitory effect on in vitro translation.  相似文献   

16.
    
The crystal structure of NADP‐dependent apo‐glyceraldehyde‐3‐phosphate dehydrogenase (apo‐GAPDH) from Synechococcus PCC 7942 is reported. The crystal structure was solved by molecular replacement and refined to an R of 21.7% and Rfree of 27.5% at 2.9 Å resolution. The structural features of apo‐GAPDH are as follows. The S‐loop has an extremely flexible conformation and the sulfate ion is only taken into the classical Pi site. A structural comparison with holo‐GAPDHs indicated that the S‐loop fixation is essential in the discrimination of NADP and NAD molecules.  相似文献   

17.
    
Crystal structures of GAPDH from Palinurus versicolor complexed with two coenzyme analogues, SNAD+ and ADP‐ribose, were determined by molecular replacement and refined at medium resolution to acceptable crystallographic factors and reasonable stereochemistry. ADP‐ribose in the ADP‐ribose–GAPDH complex adopts a rather extended conformation. The interactions between ADP‐ribose and GAPDH are extensive and in a fashion dissimilar to the coenzyme NAD+. This accounts for the strong inhibiting ability of ADP‐ribose. The conformational changes induced by ADP‐ribose binding are quite different to those induced by NAD+ binding. This presumably explains the non‐cooperative behaviour of the ADP‐ribose binding. Unexpectedly, the SNAD+–GAPDH complex reveals pairwise asymmetry. The asymmetry is significant, including the SNAD+ molecule, active‐site structure and domain motion induced by the coenzyme analogue. In the yellow or red subunits [nomenclature of subunits is as in Buehner et al. (1974). J. Mol. Biol. 90 , 25–49], SNAD+ binds similarly, as does NAD+ in holo‐GAPDH. While, in the green or blue subunit, the SNAD+ binds in a non‐productive manner, resulting in a disordered thionicotinamide ring and rearranged active‐site residues. The conformation seen in the yellow and red subunits of SNAD+–GAPDH is likely to represent the functional state of the enzyme complex in solution and thus accounts for the substrate activity of SNAD+. A novel type of domain motion is observed for the binding of the coenzyme analogues to GAPDH. The possible conformational transitions involved in the coenzyme binding and the important role of the nicotinamide group are discussed.  相似文献   

18.
    
The crystal structure of human liver glyceraldehyde‐3‐­phosphate dehydrogenase (GAPDH) has been determined. This structure represents the first moderate‐resolution (2.5 Å) and crystallographically refined (Rfree = 22.9%) human GAPDH structure. The liver GAPDH structure consists of a homotetramer, each subunit of which is bound to a nicotinamide adenine dinucleotide (NAD+) molecule. The GAPDH enzyme has glycolytic and non‐glycolytic functions, both of which are of chemotherapeutic interest. The availability of a high‐quality human GAPDH structure is a necessity for structure‐based drug design. In this study, structural differences between human liver and skeletal muscle GAPDHs are reported in order to understand how these two enzymes might respond to anti‐trypanosomatid GAPDH inhibitors.  相似文献   

19.
    
The association of glycolytic enzymes with F-actin is proposed to be one mechanism by which these enzymes are compartmentalized, and, as a result, may possibly play important roles for: regulation of the glycolytic pathway, potential substrate channeling, and increasing glycolytic flux. Historically, in vitro experiments have shown that many enzyme/actin interactions are dependent on ionic strength. Herein, Brownian dynamics (BD) examines how ionic strength impacts the energetics of the association of F-actin with the glycolytic enzymes: lactate dehydrogenase (LDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), fructose-1,6-bisphosphate aldolase (aldolase), and triose phosphate isomerase (TPI). The BD simulations are steered by electrostatics calculated by Poisson-Boltzmann theory. The BD results confirm experimental observations that the degree of association diminishes as ionic strength increases but also suggest that these interactions are significant, at physiological ionic strengths. Furthermore, BD agrees with experiments that muscle LDH, aldolase, and GAPDH interact significantly with F-actin whereas TPI does not. BD indicates similarities in binding regions for aldolase and LDH among the different species investigated. Furthermore, the residues responsible for salt bridge formation in stable complexes persist as ionic strength increases. This suggests the importance of the residues determined for these binary complexes and specificity of the interactions. That these interactions are conserved across species, and there appears to be a general trend among the enzymes, support the importance of these enzyme-F-actin interactions in creating initial complexes critical for compartmentation.  相似文献   

20.
    
GAPDH (d ‐glyceraldehyde‐3‐phosphate dehydrogenase) is a multifunctional protein that is a target for the design of antitrypanosomatid and anti‐apoptosis drugs. Here, the first high‐resolution (1.75 Å) structure of a human GAPDH is reported. The structure shows that the intersubunit selectivity cleft that has been leveraged in the design of antitrypanosomatid compounds is closed in human GAPDH. Modeling of an anti‐trypanosomatid GAPDH inhibitor in the human GAPDH active site provides insights into the basis for the observed selectivity of this class of inhibitor. Moreover, the high‐resolution data reveal a new feature of the cleft: water‐mediated intersubunit hydrogen bonds that assist closure of the cleft in the human enzyme. The structure is used in a computational ligand‐docking study of the small‐molecule compound CGP‐3466, which inhibits apoptosis by preventing nuclear accumulation of GAPDH. Plausible binding sites are identified in the adenosine pocket of the NAD+‐binding site and in a hydrophobic channel located in the center of the tetramer near the intersection of the three molecular twofold axes. The structure is also used to build a qualitative model of the complex between GAPDH and the E3 ubiquitin ligase Siah1. The model suggests that the convex surface near GAPDH Lys227 interacts with a large shallow groove of the Siah1 dimer. These results are discussed in the context of the recently discovered NO–S‐nitrosylation–GAPDH–Siah1 apoptosis cascade.  相似文献   

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