ABSTRACT
Myo-inositol-1-phosphate synthase (MIPS) catalyzes the NAD+-dependent isomerization of glucose-6-phosphate (G6P) into inositol-1-phosphate (IMP), controlling the rate-limiting step of the inositol pathway. Previous structural studies focused on the detailed molecular mechanism, neglecting large-scale conformational changes that drive the function of this 240 kDa homotetrameric complex. In this study, we identified the active, endogenous MIPS in cell extracts from the thermophilic fungus Thermochaetoides thermophila. By resolving the native structure at 2.48 Å (FSC = 0.143), we revealed a fully populated active site. Utilizing 3D variability analysis, we uncovered conformational states of MIPS, enabling us to directly visualize an order-to-disorder transition at its catalytic center. An acyclic intermediate of G6P occupied the active site in two out of the three conformational states, indicating a catalytic mechanism where electrostatic stabilization of high-energy intermediates plays a crucial role. Examination of all isomerases with known structures revealed similar fluctuations in secondary structure within their active sites. Based on these findings, we established a conformational selection model that governs substrate binding and eventually inositol availability. In particular, the ground state of MIPS demonstrates structural configurations regardless of substrate binding, a pattern observed across various isomerases. These findings contribute to the understanding of MIPS structure-based function, serving as a template for future studies targeting regulation and potential therapeutic applications.
Subject(s)
Catalytic Domain , Inositol , Myo-Inositol-1-Phosphate Synthase , Myo-Inositol-1-Phosphate Synthase/metabolism , Myo-Inositol-1-Phosphate Synthase/genetics , Myo-Inositol-1-Phosphate Synthase/chemistry , Inositol/metabolism , Inositol/chemistry , Inositol Phosphates/metabolism , Glucose-6-Phosphate/metabolism , Glucose-6-Phosphate/chemistry , Models, Molecular , Protein Conformation , Fungal Proteins/metabolism , Fungal Proteins/chemistryABSTRACT
Sugar phosphates are potential sources of carbon and phosphate for bacteria. Despite that the process of internalization of Glucose-6-Phosphate (G6P) through plasma membrane remained elusive in several bacteria. VCA0625-27, made of periplasmic ligand binding protein (PLBP) VCA0625, an atypical monomeric permease VCA0626, and a cytosolic ATPase VCA0627, recently emerged as hexose-6-phosphate uptake system of Vibrio cholerae. Here we report high resolution crystal structure of VCA0625 in G6P bound state that largely resembles AfuA of Actinobacillus pleuropneumoniae. MD simulations on VCA0625 in apo and G6P bound states unraveled an 'open to close' and swinging bi-lobal motions, which are diminished upon G6P binding. Mutagenesis followed by biochemical assays on VCA0625 underscored that R34 works as gateway to bind G6P. Although VCA0627 binds ATP, it is ATPase deficient in the absence of VCA0625 and VCA0626, which is a signature phenomenon of type-I ABC importer. Further, modeling, docking and systematic sequence analysis allowed us to envisage the existence of similar atypical type-I G6P importer with fused monomeric permease in 27 other gram-negative bacteria.
Subject(s)
ATP-Binding Cassette Transporters , Bacterial Proteins , Glucose-6-Phosphate , Vibrio cholerae , ATP-Binding Cassette Transporters/metabolism , ATP-Binding Cassette Transporters/chemistry , ATP-Binding Cassette Transporters/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Binding Sites , Crystallography, X-Ray , Glucose-6-Phosphate/metabolism , Glucose-6-Phosphate/chemistry , Models, Molecular , Molecular Dynamics Simulation , Protein Binding , Protein Conformation , Vibrio cholerae/metabolism , Vibrio cholerae/geneticsABSTRACT
Droplet formation via liquid-liquid phase separation is thought to be involved in the regulation of various biological processes, including enzymatic reactions. We investigated a glycolytic enzymatic reaction, the conversion of glucose-6-phosphate to 6-phospho-D-glucono-1,5-lactone with concomitant reduction of NADP+ to NADPH both in the absence and presence of dynamically controlled liquid droplet formation. Here, the nucleotide serves as substrate as well as the scaffold required for the formation of liquid droplets. To further expand the process parameter space, temperature and pressure dependent measurements were performed. Incorporation of the reactants in the liquid droplet phase led to a boost in enzymatic activity, which was most pronounced at medium-high pressures. The crowded environment of the droplet phase induced a marked increase of the affinity of the enzyme and substrate. An increase in turnover number in the droplet phase at high pressure contributed to a further strong increase in catalytic efficiency. Enzyme systems that are dynamically coupled to liquid condensate formation may be the key to deciphering many biochemical reactions. Expanding the process parameter space by adjusting temperature and pressure conditions can be a means to further increase the efficiency of industrial enzyme utilization and help uncover regulatory mechanisms adopted by extremophiles.
Subject(s)
Glucosephosphate Dehydrogenase , Pressure , Enzyme Activation , Gluconates/metabolism , Gluconates/chemistry , Glucose-6-Phosphate/metabolism , Glucose-6-Phosphate/chemistry , Glucosephosphate Dehydrogenase/metabolism , Glucosephosphate Dehydrogenase/chemistry , Kinetics , Lactones/chemistry , Lactones/metabolism , NADP/metabolism , NADP/chemistry , TemperatureABSTRACT
Cold-adapted enzymes maintain correct conformation at their active sites despite their intrinsically flexible structures. The psychrophilic Arctic bacterium Sphingomonas sp. PAMC 26621 has two glucose 6-phosphate dehydrogenase (G6PD) isozymes, SpG6PD1 involved in the Entner-Doudoroff pathway and SpG6PD2 in the oxidative pentose phosphate pathway. Structural modeling of SpG6PD1 showed that the hydroxyl group of Tyr177 participates in substrate binding by forming a hydrogen bond with the phosphate group of glucose 6-phosphate, whereas in SpG6PD2, a Phe residue is present in the corresponding position of Tyr177. In this study, we investigated how subtle differences in aromatic residues in the substrate-binding pocket of SpG6PD1 affect enzymatic activity and stability. Mutations of Tyr177 to Ala, His, Phe, and Trp caused increases in the rigidity of the SpG6PD1 structure. Particularly, mutants Y177F and Y177W showed increased thermal stabilities compared to wild-type (WT) but 3- and 15-fold lower catalytic efficiencies, respectively. However, mutants Y177A and Y177H became heat-labile at moderate temperatures. These results indicate that an aromatic residue (Tyr or Phe) is necessary for the substrate-binding pocket of SpG6PD1; Tyr with its hydroxyl group is preferred for enzymatic activity, whereas the more hydrophobic Phe is preferred for thermal stability. Substitutions of bulky Trp for Tyr or Phe at this position resulted in substantial loss of activity. Our study suggests that delicate adjustment of aromatic residues can regulate the activity and stability of psychrophilic G6PD isozymes involved in different metabolic pathways.
Subject(s)
Bacterial Proteins/chemistry , Glucose-6-Phosphate/chemistry , Glucosephosphate Dehydrogenase/chemistry , Phenylalanine/chemistry , Sphingomonas/chemistry , Tyrosine/chemistry , Adaptation, Physiological , Amino Acid Substitution , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , Biocatalysis , Cold Temperature , Gene Expression , Glucose-6-Phosphate/metabolism , Glucosephosphate Dehydrogenase/genetics , Glucosephosphate Dehydrogenase/metabolism , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Kinetics , Molecular Docking Simulation , Mutagenesis, Site-Directed , Phenylalanine/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Sphingomonas/enzymology , Substrate Specificity , Tryptophan/chemistry , Tryptophan/metabolism , Tyrosine/metabolismABSTRACT
A hydrophilic carbohydrate functionalized magnetic metal organic framework (Mag Zr-MOF@G6P) was synthesized via a facile one-step modification strategy for selective glycopeptide capture in virtue of hydrophilic interaction chromatography technique. The inherently hydrophilic Zr-MOF layer not only provides selective size-sieving pore structures but also offers large specific surface area to afford abundant affinity sites. Hydroxyl-rich glucose-6-phosphate was immobilized onto the Zr-MOF via a straightforward coordination manner to regulate its surface property, for the purpose of enhancing its hydrophilicity. Benefitting from the merits of Zr-MOF and glucose-6-phosphate, the as-designed composite exhibits good selectivity (the mass ratio of HRP digests to BSA digests was up to1:200) and low limit of detection (0.1 fmol µL-1) towards the recognition of glycopeptides from standard samples. More excitingly, glycopeptides in urine of healthy people and patients with kidney cancer were successfully enriched and identified by the combined liquid chromatography-mass spectrometry/mass spectrometry technology (LC-MS/MS). Further gene ontology analysis of molecular function and biological process revealed that 13 original glycoproteins of the identified glycopeptides from urine of patients significantly participate in diverse cancer-associated events, including collagen binding, immunoglobulin receptor binding, antigen binding, and complement activation process. Graphical abstract.
Subject(s)
Chromatography, Liquid/methods , Glucose-6-Phosphate/chemistry , Glycopeptides/urine , Kidney Neoplasms/urine , Metal-Organic Frameworks/chemistry , Phthalic Acids/chemistry , Tandem Mass Spectrometry/methods , Humans , Kidney Neoplasms/diagnosis , Magnetics , Urinalysis/methodsABSTRACT
Activation of phosphoenolpyruvate carboxylase (PEPC) enzymes by glucose 6-phosphate (G6P) and other phospho-sugars is of major physiological relevance. Previous kinetic, site-directed mutagenesis and crystallographic results are consistent with allosteric activation, but the existence of a G6P-allosteric site was questioned and competitive activation-in which G6P would bind to the active site eliciting the same positive homotropic effect as the substrate phosphoenolpyruvate (PEP)-was proposed. Here, we report the crystal structure of the PEPC-C4 isozyme from Zea mays with G6P well bound into the previously proposed allosteric site, unambiguously confirming its existence. To test its functionality, Asp239-which participates in a web of interactions of the protein with G6P-was changed to alanine. The D239A variant was not activated by G6P but, on the contrary, inhibited. Inhibition was also observed in the wild-type enzyme at concentrations of G6P higher than those producing activation, and probably arises from G6P binding to the active site in competition with PEP. The lower activity and cooperativity for the substrate PEP, lower activation by glycine and diminished response to malate of the D239A variant suggest that the heterotropic allosteric activation effects of free-PEP are also abolished in this variant. Together, our findings are consistent with both the existence of the G6P-allosteric site and its essentiality for the activation of PEPC enzymes by phosphorylated compounds. Furthermore, our findings suggest a central role of the G6P-allosteric site in the overall kinetics of these enzymes even in the absence of G6P or other phospho-sugars, because of its involvement in activation by free-PEP.
Subject(s)
Glucose-6-Phosphate/chemistry , Phosphoenolpyruvate Carboxylase/chemistry , Phosphoenolpyruvate/chemistry , Plant Proteins/chemistry , Zea mays/enzymology , Allosteric Regulation , Catalytic Domain , Glucose-6-Phosphate/metabolism , Kinetics , Phosphoenolpyruvate/metabolism , Phosphoenolpyruvate Carboxylase/genetics , Phosphoenolpyruvate Carboxylase/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Zea mays/geneticsABSTRACT
In general, eukaryotic glucose-6-phosphate dehydrogenases (G6PDHs) are structurally stabilized by NADP+. Here we show by spectrofluorometric analysis, thermal and urea denaturation, and trypsin proteolysis, that a different mechanism stabilizes the enzyme from Pseudomonas aeruginosa (PaG6PDH) (EC 1.1.1.363). The spectrofluorometric analysis of the emission of 8-anilino-1-naphthalenesulfonic acid (ANS) indicates that this stabilization is the result of a structural change in the enzyme caused by G6P. The similarity between the Kd values determined for the PaG6PDH-G6P complex (78.0⯱â¯7.9⯵M) and the K0.5 values determined for G6P (57.9⯱â¯2.5 and 104.5⯱â¯9.3⯵M in the NADP+- and NAD+-dependent reactions, respectively) suggests that the structural changes are the result of G6P binding to the active site of PaG6PDH. Modeling of PaG6PDH indicated the residues that potentially bind the ligand. These results and a phylogenetic analysis of the amino acid sequences of forty-four G6PDHs, suggest that the stabilization observed for PaG6PDH could be a characteristic that distinguishes this and other G6PDHs that use NAD+ and NADP+ from those that use NADP+ only or preferentially, such as those found in eukaryotes. This characteristic could be related to the metabolic roles these enzymes play in the organisms to which they belong.
Subject(s)
Glucosephosphate Dehydrogenase/metabolism , Pseudomonas aeruginosa/enzymology , Amino Acid Sequence , Anilino Naphthalenesulfonates/chemistry , Binding Sites , Catalytic Domain , Glucose-6-Phosphate/chemistry , Glucose-6-Phosphate/metabolism , Glucosephosphate Dehydrogenase/classification , Glucosephosphate Dehydrogenase/genetics , Kinetics , Molecular Dynamics Simulation , NAD/metabolism , NADP/chemistry , NADP/metabolism , Phylogeny , Protein Binding , Protein Denaturation , Protein Stability , Protein Structure, Tertiary , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purificationABSTRACT
Reducing sugars can react with 1-phenyl-3-methyl-5-pyrazolone (PMP) to form sugar-PMP derivatives, which can be detected by HPLC-UV or HPLC-DAD due to their high UV absorbance at 248â¯nm. Six different sugars were synthesized with PMP with aid of response surface methodology (RSM), by which the parameters of the synthesis were designed within temperature ranged between 60⯰C and 90⯰C, and time from 60 to 180â¯min, respectively. Consequently, optimal conditions of the glucose (Glu)-, glucosamine (GluN)-, galactose (Gal)-, glucuronic acid (GluA), galacturonic acid (GalA) and glucose-6-phosphate (G6P-PMP) reactions were determined at 71⯰C for 129â¯min, 73⯰C for 96â¯min, 70⯰C for 117â¯min, 75⯰C for 151â¯min, 76⯰C for 144â¯min, and 70⯰C for 154â¯min, respectively. Experiments demonstrated that unique functional groups and delicate differences of carbohydrates' inner pH environment could significantly influence the sugar-PMP reactions. However, sugar stereoisomers did not have remarkable impacts on the reactions.
Subject(s)
Carbohydrates/analysis , Carbohydrates/chemistry , Edaravone/chemistry , Chromatography, High Pressure Liquid , Galactose/analysis , Galactose/chemistry , Glucosamine/analysis , Glucosamine/chemistry , Glucose/analysis , Glucose/chemistry , Glucose-6-Phosphate/analysis , Glucose-6-Phosphate/chemistry , Glucuronic Acid/analysis , Glucuronic Acid/chemistry , Hexuronic Acids/analysis , Hexuronic Acids/chemistry , StereoisomerismABSTRACT
We believe that "the simpler we are, the more complete we become" is a key concept of chemical sensing systems. In this work, a "turn-on" fluorescence chemosensor array relying on only two self-assembled molecular chemosensors with ability of both qualitative and quantitative detection of phosphorylated saccharides has been developed. The easy-to-prepare chemosensor array was fabricated by in situ mixing of off-the-shelf reagents (esculetin, 4-methylesculetin, and 3-nitrophenylboronic acid). The fluorescence-based saccharide sensing system was carried out using indicator displacement assay accompanied by photoinduced electron transfer (PeT) under various pH conditions. The simultaneous recognition of 14 types of saccharides including glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P) was achieved with a successful classification rate of 100%. We also succeeded in the quantitative analysis of a mixture of glucose (Glc), as an original substrate, G6P and F6P, as enzymatic products in pseudoglycolysis pathway. Finally, levels of Glc and F6P in human induced pluripotent stem (hiPS) cells were indirectly monitored by using our proposed chemosensor array. Glc and F6P in supernatants of hiPS cells were classified by linear discriminant analysis as a pattern recognition model and the observed clusters represent the activity of hiPS cells. The results show the high accuracy of the proposed chemosensor array in detection of phosphorylated and similarly modified saccharides.
Subject(s)
Biosensing Techniques/methods , Boronic Acids/chemistry , Fructosephosphates/analysis , Glucose-6-Phosphate/analysis , Glucose/analysis , Induced Pluripotent Stem Cells/metabolism , Cells, Cultured , Fluorescence , Fructosephosphates/chemistry , Glucose/chemistry , Glucose-6-Phosphate/chemistry , Humans , Induced Pluripotent Stem Cells/cytology , PhosphorylationABSTRACT
A large number of mutations causing PMM2-CDG, which is the most frequent disorder of glycosylation, destabilize phosphomannomutase2. We looked for a pharmacological chaperone to cure PMM2-CDG, starting from the structure of a natural ligand of phosphomannomutase2, α-glucose-1,6-bisphosphate. The compound, ß-glucose-1,6-bisphosphate, was synthesized and characterized via 31P-NMR. ß-glucose-1,6-bisphosphate binds its target enzyme in silico. The binding induces a large conformational change that was predicted by the program PELE and validated in vitro by limited proteolysis. The ability of the compound to stabilize wild type phosphomannomutase2, as well as frequently encountered pathogenic mutants, was measured using thermal shift assay. ß-glucose-1,6-bisphosphate is relatively resistant to the enzyme that specifically hydrolyses natural esose-bisphosphates.
Subject(s)
Congenital Disorders of Glycosylation/genetics , Congenital Disorders of Glycosylation/metabolism , Glucose-6-Phosphate/analogs & derivatives , Mutation , Phosphotransferases (Phosphomutases)/deficiency , Congenital Disorders of Glycosylation/drug therapy , Glucose-6-Phosphate/chemistry , Glucose-6-Phosphate/metabolism , Glucose-6-Phosphate/pharmacology , Humans , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Conformation , Phosphotransferases (Phosphomutases)/genetics , Protein BindingABSTRACT
Back scatter interferometry (BSI) is a sensitive method for detecting changes in the bulk refractive index of a solution in a microfluidic system. Here we demonstrate that BSI can be used to directly detect enzymatic reactions and, for the first time, derive kinetic parameters. While many methods in biomedical assays rely on detectable biproducts to produce a signal, direct detection is possible if the substrate or the product exert distinct differences in their specific refractive index so that the total refractive index changes during the enzymatic reaction. In this study, both the conversion of glucose to glucose-6-phosphate, catalyzed by hexokinase, and the conversion of adenosine-triphosphate to adenosine di-phosphate and mono-phosphate, catalyzed by apyrase, were monitored by BSI. When adding hexokinase to glucose solutions containing adenosine-triphosphate, the conversion can be directly followed by BSI, which shows the increasing refractive index and a final plateau corresponding to the particular concentration. From the initial reaction velocities, KM was found to be 0.33 mM using Michaelisâ»Menten kinetics. The experiments with apyrase indicate that the refractive index also depends on the presence of various ions that must be taken into account when using this technique. This study clearly demonstrates that measuring changes in the refractive index can be used for the direct determination of substrate concentrations and enzyme kinetics.
Subject(s)
Adenosine Triphosphate/chemistry , Catalysis , Hexokinase/chemistry , Interferometry/methods , Glucose/chemistry , Glucose-6-Phosphate/chemistry , Kinetics , Microfluidics/methods , Refractometry/methods , Solutions/chemistryABSTRACT
Female Aedes aegypti mosquitoes are vectors of arboviruses that cause diseases of public health significance. The discovery of new metabolic targets is crucial for improving mosquito control strategies. We recently demonstrated that glucose oxidation supports ammonia detoxification in A. aegypti. Pyruvate kinase (PK, EC 2.7.1.40) catalyzes the last step of the glycolytic pathway. In most organisms, one or more allosteric effectors control PK activity. However, the kinetic properties and structure of PK in mosquitoes have not been previously reported. In this study, two alternatively spliced mRNA variants (AaPK1 and AaPK2) that code for PKs were identified in the A. aegypti genome. The AaPK1 mRNA variant, which encodes a 529 amino acid protein with an estimated molecular weight of â¼57â¯kDa, was cloned. The protein was expressed in Escherichia coli and purified. The AaPK1 kinetic properties were identified. The recombinant protein was also crystallized and its 3D structure determined. We found that alanine, glutamine, proline, serine and fructose-1-phosphate displayed a classic allosteric activation on AaPK1. Ribulose-5-phosphate acted as an allosteric inhibitor of AaPK1 but its inhibitory effect was reversed by alanine, glutamine, proline and serine. Additionally, the allosteric activation of AaPK1 by amino acids was weakened by fructose-1,6-bisphosphate, whereas the allosteric activation of AaPK1 by alanine and serine was diminished by glucose-6-phosphate. The AaPK1 structure shows the presence of fructose-1,6-bisphosphate in the allosteric site. Together, our results reveal that specific amino acids and phosphorylated sugars tightly regulate conformational dynamics and catalytic changes of AaPK1. The distinctive AaPK1 allosteric properties support a complex role for this enzyme within mosquito metabolism.
Subject(s)
Aedes/enzymology , Fructosediphosphates/chemistry , Glucose-6-Phosphate/chemistry , Insect Proteins/chemistry , Pyruvate Kinase/chemistry , Aedes/genetics , Allosteric Regulation/physiology , Alternative Splicing/physiology , Animals , Female , Fructosediphosphates/metabolism , Gene Expression Regulation, Enzymologic/physiology , Glucose-6-Phosphate/metabolism , Insect Proteins/biosynthesis , Insect Proteins/genetics , Kinetics , Protein Domains , Pyruvate Kinase/genetics , Pyruvate Kinase/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
A series of novel pyridine and fused pyridine derivatives have been prepared starting from 6-(3,4-dimethylphenyl)-2-hydrazinyl-4-(thiophen-2-yl)-pyridine-3-carbonitrile 1 which on treatment with appropriate formic acid, acetic acid/ acetic anhydride, benzoyl chloride and/or carbon disulfide afforded the corresponding triazolopyridine derivatives 2â»5. Also, treatment of hydrazide 1 with diethyloxalate, chloroacetyl chloride, chloroacetic acid and/or 1,2-dichloroethane yielded the corresponding pyridotriazine derivatives 7â»10. Further transformation of compound 1 with a different active methylene group, namely acetyl acetone, diethylmalonate, ethyl cyanoacetate, ethyl benzoylacetate and/or ethyl acetoacetate, produced the pyridineâ»pyrazole hybrid derivatives 11â»15. These newly synthesized compounds (1â»15) were subjected to in silico molecular docking screenings towards GlcN-6-P synthase as the target protein. The results revealed moderate to good binding energies of the ligands on the target protein. All the newly prepared products exhibited antimicrobial and antioxidant activity.
Subject(s)
Anti-Bacterial Agents/chemical synthesis , Anti-Infective Agents/chemical synthesis , Pyrazoles/chemical synthesis , Pyridines/chemistry , Acetic Acid/chemical synthesis , Acetic Acid/chemistry , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Anti-Infective Agents/chemistry , Anti-Infective Agents/pharmacology , Benzoates/chemistry , Carbon Disulfide/chemical synthesis , Carbon Disulfide/chemistry , Formates/chemical synthesis , Formates/chemistry , Glucosamine/analogs & derivatives , Glucosamine/chemistry , Glucose-6-Phosphate/analogs & derivatives , Glucose-6-Phosphate/chemistry , Microbial Sensitivity Tests , Molecular Docking Simulation , Pyrazoles/chemistry , Pyrazoles/pharmacology , Pyridines/chemical synthesis , Pyridines/pharmacology , Structure-Activity RelationshipABSTRACT
N-acetylphosphoglucosamine mutase (AGM1) is a key component of the hexosamine biosynthetic pathway that produces UDP-GlcNAc, an essential precursor for a wide range of glycans in eukaryotes. AGM belongs to the α-d-phosphohexomutase metalloenzyme superfamily and catalyzes the interconversion of N-acetylglucosamine-6-phosphate (GlcNAc-6P) to N-acetylglucosamine-1-phosphate (GlcNAc-1P) through N-acetylglucosamine-1,6-bisphosphate (GlcNAc-1,6-bisP) as the catalytic intermediate. Although there is an understanding of the phosphoserine-dependent catalytic mechanism at enzymatic and structural level, the identity of the requisite catalytic base in AGM1/phosphoglucomutases is as yet unknown. Here, we present crystal structures of a Michaelis complex of AGM1 with GlcNAc-6P and Mg2+, and a complex of the inactive Ser69Ala mutant together with glucose-1,6-bisphosphate (Glc-1,6-bisP) that represents key snapshots along the reaction co-ordinate. Together with mutagenesis, these structures reveal that the phosphate group of the hexose-1,6-bisP intermediate may act as the catalytic base.
Subject(s)
Acetylglucosamine/analogs & derivatives , Aspergillus fumigatus/enzymology , Fungal Proteins/chemistry , Glucose-6-Phosphate/analogs & derivatives , Phosphoglucomutase/chemistry , Acetylglucosamine/chemistry , Acetylglucosamine/metabolism , Catalysis , Fungal Proteins/metabolism , Glucose-6-Phosphate/chemistry , Glucose-6-Phosphate/metabolism , Phosphoglucomutase/metabolismABSTRACT
As one of the most important post-translational modifications (PTMs) of peptidome, glycopeptidome is closely related to serious disease, especially to cancer. In order to specifically discover and analyze glycopeptidome biomarkers for clinical diagnosis of cancer on early-stage, it is crucial to develop efficient technique to analyze low-abundance of endogenous glycopeptides in biological samples. In this report, a hydrophilic probe in mesoporous pore (denoted as Fe3O4@mSiO2@G6P) was designed and prepared. By taking advantage of the excellent hydrophilicity and size-exclusion ability, we applied Fe3O4@mSiO2@G6P to capture glycopeptides from both horseradish peroxidase (HRP) and immunoglobulin (IgG) digests successfully. Moreover, a total of 39 and 25 endogenous glycopeptides were identified from healthy saliva and gastric saliva, respectively, indicating the great potential of this probe for the exploration of glycopeptidome biomarkers.
Subject(s)
Glycopeptides/analysis , Hydrophobic and Hydrophilic Interactions , Molecular Probes/chemistry , Proteome/analysis , Biomarkers/analysis , Biomarkers/chemistry , Ferric Compounds/chemistry , Glucose-6-Phosphate/chemistry , Glycopeptides/chemistry , Horseradish Peroxidase/chemistry , Humans , Immunoglobulin G/chemistry , Magnetite Nanoparticles/chemistry , Molecular Probes/chemical synthesis , Porosity , Proteome/chemistry , Saliva/chemistry , Silicon Dioxide/chemistryABSTRACT
The high proliferation rate of tumor cells demands high energy and metabolites that are sustained by a high glycolytic flux known as the 'Warburg effect'. The activation and further metabolism of glucose is initiated by hexokinase, a focal point of metabolic regulation. The human hexokinase 2 (HK2) is overexpressed in all aggressive tumors and predominantly found on the outer mitochondrial membrane, where interactions through its N-terminus initiates and maintains tumorigenesis. Here, we report the structure of HK2 in complex with glucose and glucose-6-phosphate (G6P). Structural and biochemical characterization of the mitochondrial conformation reveals higher conformational stability and slow protein unfolding rate (ku) compared with the cytosolic conformation. Despite the active site similarity of all human hexokinases, the N-domain of HK2 is catalytically active but not in hexokinase 1 and 3. Helix-α13 that protrudes out of the N-domain to link it to the C-domain of HK2 is found to be important in maintaining the catalytic activity of the N-half. In addition, the N-domain of HK2 regulates the stability of the whole enzyme in contrast with the C-domain. Glucose binding enhanced the stability of the wild-type (WT) enzyme and the single mutant D657A of the C-domain, but it did not increase the stability of the D209A mutant of the N-domain. The interaction of HK2 with the mitochondria through its N-half is proposed to facilitate higher stability on the mitochondria. The identification of structural and biochemical differences between HK2 and other human hexokinase isozymes could potentially be used in the development of new anticancer therapies.
Subject(s)
Glucose-6-Phosphate/chemistry , Glucose/metabolism , Hexokinase/chemistry , Mitochondria/enzymology , Mitochondrial Membranes/enzymology , Catalysis , Catalytic Domain , Glucose/chemistry , Hexokinase/genetics , Humans , Isoenzymes/chemistry , Isoenzymes/metabolism , Kinetics , Molecular Conformation , Mutation , Protein Conformation , ThermodynamicsABSTRACT
F420-dependent glucose-6-phosphate dehydrogenase (FGD) is involved in the committed step of the pentose phosphate pathway within mycobacteria, where it catalyzes the reaction between glucose-6-phosphate (G6P) and the F420 cofactor to yield 6-phosphogluconolactone and the reduced cofactor, F420H2. Here, we aim to probe the FGD reaction mechanism using dead-end inhibition experiments, as well as solvent and substrate deuterium isotope effects studies. The dead-end inhibition studies performed using citrate as the inhibitor revealed competitive and uncompetitive inhibition patterns for G6P and F420 respectively, thus suggesting a mechanism of ordered addition of substrates in which the F420 cofactor must first bind to FGD before G6P binding. The solvent deuterium isotope effects studies yielded normal solvent kinetic isotope effects (SKIE) on kcat and kcat/Km for both G6P and F420. The proton inventory data yielded a fractionation factor of 0.37, suggesting that the single proton responsible for the observed SKIE is likely donated by Glu109 and protonates the cofactor at position N1. The steady state substrate deuterium isotope effects studies using G6P and G6P-d1 yielded KIE of 1.1 for both kcat and kcat/Km, while the pre-steady state KIE on kobs was 1.4. Because the hydride transferred to C5 of F420 was the one targeted for isotopic substitution, these KIE values provide further evidence to support our previous findings that hydride transfer is likely not rate-limiting in the FGD reaction.
Subject(s)
Bacterial Proteins/chemistry , Deuterium/chemistry , Glucose-6-Phosphate/chemistry , Glucosephosphate Dehydrogenase/chemistry , Mycobacterium tuberculosis/enzymology , Citric Acid/chemistry , Deuterium Exchange Measurement/methodsABSTRACT
In a previous study the full-length open reading frame of the Arabian camel, Camelus dromedarius liver cytosolic glucose-6-phosphate dehydrogenase (G6PD) cDNA was determined using reverse transcription polymerase chain reaction. The C. dromedarius cDNA was found to be 1545 nucleotides (accession number JN098421) that encodes a protein of 515 amino acids residues. In the present study, C. dromedarius recombinant G6PD was heterologously overexpressed in Escherichia coli BL21 (DE3) pLysS and purified by immobilized metal affinity fast protein liquid chromatography (FPLC) in a single step. The purity and molecular weight of the enzyme were analyzed on SDS-PAGE and the purified enzyme showed a single band on the gel with a molecular weight of 63.0 KDa. The specific activity was determined to be 2000 EU/mg protein. The optimum temperature and pH were found to be 60 °C and 7.4, respectively. The isoelectric point (pI) for the purified G6PD was determined to be 6.4. The apparent Km values for the two substrates NADP+ and G6P were found to be 23.2 µM and 66.7 µM, respectively. The far-UV circular dichroism (CD) spectra of G6PD showed that it has two minima at 208 and 222 nm as well as maxima at 193 nm which is characteristic of high content of α-helix. Moreover, the far-UV CD spectra of the G6PD in the presence or absence of NADP+ were nearly identical.
Subject(s)
Glucose-6-Phosphate/chemistry , Glucosephosphate Dehydrogenase/metabolism , NADP/chemistry , Plasmids/chemistry , Animals , Camelus , Cloning, Molecular , Enzyme Assays , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Glucosephosphate Dehydrogenase/genetics , Hydrogen-Ion Concentration , Isoelectric Point , Kinetics , Liver/chemistry , Liver/enzymology , Molecular Weight , Plasmids/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate SpecificityABSTRACT
Glucosamine-6-phosphate synthase (EC 2.6.1.16) is responsible for catalysis of the first and practically irreversible step in hexosamine metabolism. The final product of this pathway, uridine 5' diphospho N-acetyl-d-glucosamine (UDP-GlcNAc), is an essential substrate for assembly of bacterial and fungal cell walls. Moreover, the enzyme is involved in phenomenon of hexosamine induced insulin resistance in type II diabetes, which makes of it a potential target for anti-fungal, anti-bacterial and anti-diabetic therapy. The crystal structure of isomerase domain from human pathogenic fungus Candida albicans has been solved recently but it doesn't reveal the molecular mechanism details of inhibition taking place under UDP-GlcNAc influence, the unique feature of eukaryotic enzyme. The following study is a continuation of the previous research based on comparative molecular dynamics simulations of the structures with and without the enzyme's physiological inhibitor (UDP-GlcNAc) bound. The models used for this study included fructose-6-phosphate, one of the enzyme's substrates in its binding pocket. The simulation results studies demonstrated differences in mobility of the compared structures. Some amino acid residues were determined, for which flexibility is evidently different between the models. Importantly, it has been confirmed that the most fixed residues are related to the inhibitor binding process and to the catalysis reaction. The obtained results constitute an important step towards understanding of the inhibition that GlcN-6-P synthase is subjected by UDP-GlcNAc molecule.
Subject(s)
Acetylglucosamine/chemistry , Candida albicans/enzymology , Fructosephosphates/chemistry , Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)/chemistry , Amino Acids/chemistry , Candida albicans/chemistry , Catalysis , Glucosamine/analogs & derivatives , Glucosamine/chemistry , Glucose-6-Phosphate/analogs & derivatives , Glucose-6-Phosphate/chemistry , Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)/antagonists & inhibitors , Humans , Kinetics , Molecular Dynamics Simulation , Protein Binding , Protein ConformationABSTRACT
Developing cost-effective approaches based on hydrophilic interaction liquid chromatography (HILIC) has been the main tendency for low-abundance glycopeptides capture before LC-MS/MS analysis. Carbohydrates with outstanding biocompatibility and hydrophilicity are ubiquitous in the kingdoms of animal and plant and could be a wonderful choice as functional groups for glycopeptides enrichment. In this work, glucose-6-phosphate, as one of the indispensable cogs in pivotal metabolic wheels of life, was chosen as functionalized groups to be grafted onto the surface of Fe3O4 microspheres via one-step surface fabrication strategy. The acquired hydrophilic Fe3O4@G6P microspheres showed superior enrichment performance for glycopeptides with high sensitivity (0.5 fmol/µL) and high selectivity (1:100) and good repeatability (10 times at least). Furthermore, the Fe3O4@G6P microspheres also exhibited enrichment ability for glycopeptides in different biosamples. A total of 243 glycopeptides assigned to 92 glycoproteins and 183 glycopeptides corresponding to 74 different glycoproteins was identified from merely 2 µL of serum and saliva, respectively.