Affinity labelling of the NADP+-binding site of glucose 6-phosphate dehydrogenase from Candida utilis.
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ABSTRACT:
SUBMITTER: Bellini T
PROVIDER: S-EPMC1161558 | biostudies-other | 1979 Nov
REPOSITORIES: biostudies-other
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Project description:Human glucose 6-phosphate dehydrogenase, purified after overexpression in E. coli, was shown to contain one molecule/subunit of acid-extractable "structural" NADP+ and no NADPH. This tightly bound NADP+ was reduced by G6P, presumably following migration to the catalytic site. Gel-filtration yielded apoenzyme, devoid of bound NADP+ but, surprisingly, still fully active. Mr of the main component of "stripped" enzyme by gel filtration was approximately 100,000, suggesting a dimeric apoenzyme (subunit Mr = 59,000). Holoenzyme also contained tetramer molecules and, at high protein concentration, a dynamic equilibrium gave an apparent intermediate Mr of 150 kDa. Fluorescence titration of the stripped enzyme gave the K d for structural NADP+ as 37 nM, 200-fold lower than for "catalytic" NADP+. Structural NADP+ quenches 91% of protein fluorescence. At 37 degrees C, stripped enzyme, much less stable than holoenzyme, inactivated irreversibly within 2 d. Inactivation at 4 degrees C was partially reversed at room temperature, especially with added NADP+. Apoenzyme was immediately active, without any visible lag, in rapid-reaction studies. Human G6PD thus forms active dimer without structural NADP+. Apparently, the true role of the second, tightly bound NADP+ is to secure long-term stability. This fits the clinical pattern, G6PD deficiency affecting the long-lived non-nucleate erythrocyte. The Kd values for two class I mutants, G488S and G488V, were 273 nM and 480 nM, respectively (seven- and 13-fold elevated), matching the structural prediction of weakened structural NADP+ binding, which would explain decreased stability and consequent disease. Preparation of native apoenzyme and measurement of Kd constant for structural NADP+ will now allow quantitative assessment of this defect in clinical G6PD mutations.
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Project description:Human glucose-6-phosphate dehydrogenase (G6PD) is the main cellular source of NADPH, and thus plays a key role in maintaining reduced glutathione to protect cells from oxidative stress disorders such as hemolytic anemia. G6PD is a multimeric enzyme that uses the cofactors β-D-glucose 6-phosphate (G6P) and "catalytic" NADP+ (NADP+c), as well as a "structural" NADP+ (NADP+s) located ∼25 Å from the active site, to generate NADPH. While X-ray crystallographic and biochemical studies have revealed a role for NADP+s in maintaining the catalytic activity by stabilizing the multimeric G6PD conformation, other potential roles for NADP+s have not been evaluated. Here, we determined the high resolution cryo-electron microscopy structures of human wild-type G6PD in the absence of bound ligands and a catalytic G6PD-D200N mutant bound to NADP+c and NADP+s in the absence or presence of G6P. A comparison of these structures, together with previously reported structures, reveals that the unliganded human G6PD forms a mixture of dimers and tetramers with similar overall folds, and binding of NADP+s induces a structural ordering of a C-terminal extension region and allosterically regulates G6P binding and catalysis. These studies have implications for understanding G6PD deficiencies and for therapy of G6PD-mediated disorders.
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Project description: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 A 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.
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Project description:The ability of the reactive dichlorotriazine dye Vilmafix Blue A-R (VBAR) to act as an affinity label for bovine heart L-malate dehydrogenase (MDH) was studied. VBAR binds specifically and irreversibly to MDH (k3 0.16 min-1; KD 14.4 microM). The inactivation of the NADH-dependent enzyme by VBAR is competitively inhibited by NAD+, NADH and ADP. Quantitatively inhibited MDH contained approx. 1 mol of dye per mol of active site. The inhibition is irreversible and activity cannot be recovered either on incubation with 10 mM NAD+, 10 mM NADH or 10 mM ADP, or by extensive dialysis or gel-filtration chromatography. Data obtained from high-performance gel-filtration chromatography and analysed by Scatchard plot suggested the presence of two coenzyme-binding sites per MDH dimer. Tryptic digestion of VBAR-labelled MDH followed by reverse-phase HPLC analysis revealed one VBAR-labelled peptide. It appears that each subunit features the same peptide bearing the modifying residue involved in MDH labelling. The pKa of the modifying residue is 8.05. Both total acid hydrolysis of VBAR-labelled MDH followed by HPLC and TLC analysis, and molecular-modelling studies suggest that the modifying residue is Lys-81 and/or Lys-217.
| S-EPMC1217251 | biostudies-other
Project description:AimTo characterize phosphorylation of human glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and mobility of GAPDH in cancer cells treated with chemotherapeutic agents.MethodsWe used proteomics analysis to detect and characterize phosphorylation sites within human GAPDH. Site-specific mutagenesis and alanine scanning was then performed to evaluate functional significance of phosphorylation sites in the GAPDH polypeptide chain. Enzymatic properties of mutated GAPDH variants were assessed using kinetic studies. Intranuclear dynamics parameters (diffusion coefficient and the immobile fraction) were estimated using fluorescence recovery after photobleaching (FRAP) experiments and confocal microscopy. Molecular modeling experiments were performed to estimate the effects of mutations on NAD(+) cofactor binding.ResultsUsing MALDI-TOF analysis, we identified novel phosphorylation sites within the NAD(+) binding center of GAPDH at Y94, S98, and T99. Using polyclonal antibody specific to phospho-T99-containing peptide within GAPDH, we demonstrated accumulation of phospho-T99-GAPDH in the nuclear fractions of A549, HCT116, and SW48 cancer cells after cytotoxic stress. We performed site-mutagenesis, and estimated enzymatic properties, intranuclear distribution, and intranuclear mobility of GAPDH mutated variants. Site-mutagenesis at positions S98 and T99 in the NAD(+) binding center reduced enzymatic activity of GAPDH due to decreased affinity to NAD(+) (Km = 741 ± 257 μmol/L in T99I vs 57 ± 11.1 µmol/L in wild type GAPDH. Molecular modeling experiments revealed the effect of mutations on NAD(+) binding with GAPDH. FRAP (fluorescence recovery after photo bleaching) analysis showed that mutations in NAD(+) binding center of GAPDH abrogated its intranuclear interactions.ConclusionOur results suggest an important functional role of phosphorylated amino acids in the NAD(+) binding center in GAPDH interactions with its intranuclear partners.
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Project description:In immunogold double-labeling of pea leaf thin sections with antibodies raised against ferredoxin-NADP reductase (EC 1.18.1.2, FNR) and antibodies directed against the A or B subunits of the NADP-linked glyceraldehyde-3-P dehydrogenase (GAPD) (EC 1.2.1.13), many small and large gold particles were found together over the chloroplasts. Nearest neighbor analysis of the distribution of the gold particles indicates that FNR and the NADP-linked GAPD are co-localized, in situ. This suggests that FNR might carry FADH2 or NADPH from the thylakoid membrane to GAPD, or that ferredoxin might carry electrons to FNR co-localized with GAPD in the stroma. Crystal structures of the spinach enzymes are available. When they are docked computationally, the proteins appear, as modeled, to be able to form at least two different complexes. One involves a single GAPD monomer and an FNR monomer (or dimer). The amino acid residues located at the putative interface are highly conserved on the chloroplastic forms of both enzymes. The other potential complex involves the GAPD A2B2 tetramer and an FNR monomer (or dimer). The interface residues are conserved in this model as well. Ferredoxin is able to interact with FNR in either complex.
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Project description:The available resources of Streptomyces represent a valuable repository of bioactive natural products that warrant exploration. Streptomyces albulus is primarily utilized in the industrial synthesis of ε-poly-L-lysine (ε-PL). In this study, the NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (GapN) from Streptococcus mutans was heterologously expressed in S. albulus CICC11022, leading to elevated intracellular NADPH levels and reduced NADH and ATP concentrations. The resulting perturbation of S. albulus metabolism was comprehensively analyzed using transcriptomic and metabolomic methodologies. A decrease in production of ε-PL was observed. The expression of gapN significantly impacted on 23 gene clusters responsible for the biosynthesis of secondary metabolites. A comprehensive analysis revealed a total of 21 metabolites exhibiting elevated levels both intracellularly and extracellularly in the gapN expressing strain compared to those in the control strain. These findings underscore the potential of S. albulus to generate diverse bioactive natural products, thus offering valuable insights for the utilization of known Streptomyces resources through genetic manipulation.
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Project description:Glucose-6-phosphate dehydrogenase (G6PDH) plays a housekeeping role in cell metabolism by generating reducing power (NADPH) and fueling the production of nucleotide precursors (ribose-5-phosphate). Based on its indispensability for pathogenic parasites from the genus Trypanosoma, G6PDH is considered a drug target candidate. Several steroid-like scaffolds were previously reported to target the activity of G6PDH. Epiandrosterone (EA) is an uncompetitive inhibitor of trypanosomal G6PDH for which its binding site to the enzyme remains unknown. Molecular simulation studies with the structure of Trypanosoma cruzi G6PDH revealed that EA binds in a pocket close to the G6P binding-site and protrudes into the active site blocking the interaction between substrates and hence catalysis. Site directed mutagenesis revealed the important steroid-stabilizing effect of residues (L80, K83 and K84) located on helix ?-1 of T. cruzi G6PDH. The higher affinity and potency of 16?-Br EA by T. cruzi G6PDH is explained by the formation of a halogen bond with the hydrogen from the terminal amide of the NADP+-nicotinamide. At variance with the human enzyme, the inclusion of a 21-hydroxypregnane-20-one moiety to a 3?-substituted steroid is detrimental for T. cruzi G6PDH inhibition. The species-specificity of certain steroid derivatives towards the parasite G6PDH and the corresponding biochemically validated binding models disclosed in this work may prove valuable for the development of selective inhibitors against the pathogen's enzyme.
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Project description:Fis (factor for inversion stimulation) is a nucleoid-associated protein in Escherichia coli and other bacteria that stimulates certain site-specific DNA recombination events, alters DNA topology, and serves as a global gene regulator. DNA binding is central to the functions of Fis and involves a helix-turn-helix DNA binding motif located in the carboxy-terminal region. Specific DNA binding is observed at a number of sites exhibiting poorly related sequences. Such interactions require four critical base pairs positioned -7, -3, +3, and +7 nucleotides relative to the central nucleotide of a 15-bp core-binding site. To further understand how Fis interacts with DNA, we identified the positions of 14 DNA phosphates (based on ethylation interference assays) that are required for Fis binding. These are the 5' phosphates of the nucleotides at positions -8, -7, -6, +1, +2, +3, and +4 relative to the central nucleotide on both DNA strands. Another five phosphates located in the flanking regions from positions +10 through +14 can serve as additional contact sites. Using a combination of biochemical approaches and various mutant Fis proteins, we probed possible interactions between several key Fis residues and DNA bases or phosphates within a high-affinity binding site. We provide evidence in support of interactions between the R85 Fis residue and a highly conserved guanine at position -7 and between T87 and the critical base pairs at -3 and +3. In addition, we present evidence in support of interactions between N84 and the phosphate 5' to the base at +4, between R89 and the -7 phosphate, between T87 and the +3 and +4 phosphates, and between K90 and the +3 phosphate. This work provides functional evidence for some of the most critical interactions between Fis and DNA required for a high binding affinity and demonstrates the large contribution made by numerous phosphates to the stability of the Fis-DNA complex.
| S-EPMC2517053 | biostudies-literature
Project description:Among the determinants contributing to the pathogenesis of asthma, antioxidant genetic factors play a leading role. Glucose-6-phosphate dehydrogenase (G6PD) is an enzyme that is competent to detoxify free radicals. Although a relationship between G6PD deficiency and asthma has been previously reported, the literature is still scanty. In this study, we test this hypothesis in a large cohort of patients from Sardinia, Italy. A retrospective case-control study was performed using data from 11,829 clinical records of outpatients referred to a teaching hospital for a medical visit. In total, 455 cases (asthma-positive) and 11,374 controls (asthma-negative) were compared for G6PD status using multivariable analysis, adjusting for all covariates. Overall, G6PD deficiency was detected in 11.2% of study participants and was associated with an increased risk of asthma (odds ratio (OR) 1.63; 95% confidence interval (CI) 1.27-2.10). Additional variables significantly associated with asthma were female sex (OR 1.66; 95% CI 1.34-2.06), overweight/obesity (OR 1.56; 95% CI 1.27-1.92), smoking (OR 1.44; 95% CI 1.449-3.963), and high socioeconomic status (OR 1.40; 95% CI 1.16-1.70), whereas age was inversely related with asthma (OR 0.49; 95% CI 0.39-0.61). Our study shows that G6PD deficiency is an independent risk for asthma. These findings suggest that G6PD should be assessed in asthmatic patients for better risk stratification.
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