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Amoxicillin Haptenation of α-Enolase is Modulated by Active Site Occupancy and Acetylation.

ABSTRACT: Allergic reactions to antibiotics are a major concern in the clinic. ß-lactam antibiotics are the class most frequently reported to cause hypersensitivity reactions. One of the mechanisms involved in this outcome is the modification of proteins by covalent binding of the drug (haptenation). Hence, interest in identifying the corresponding serum and cellular protein targets arises. Importantly, haptenation susceptibility and extent can be modulated by the context, including factors affecting protein conformation or the occurrence of other posttranslational modifications. We previously identified the glycolytic enzyme α-enolase as a target for haptenation by amoxicillin, both in cells and in the extracellular milieu. Here, we performed an in vitro study to analyze amoxicillin haptenation of α-enolase using gel-based and activity assays. Moreover, the possible interplay or interference between amoxicillin haptenation and acetylation of α-enolase was studied in 1D- and 2D-gels that showed decreased haptenation and displacement of the haptenation signal to lower pI spots after chemical acetylation of the protein, respectively. In addition, the peptide containing lysine 239 was identified by mass spectrometry as the amoxicillin target sequence on α-enolase, thus suggesting a selective haptenation under our conditions. The putative amoxicillin binding site and the surrounding interactions were investigated using the α-enolase crystal structure and molecular docking. Altogether, the results obtained provide the basis for the design of novel diagnostic tools or approaches in the study of amoxicillin-induced allergic reactions.

SUBMITTER: Gonzalez-Morena JM

PROVIDER: S-EPMC8793629 | biostudies-literature |

REPOSITORIES: biostudies-literature

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AMOXICILLIN HAPTENATION OF α-ENOLASE

Project description:Allergic reactions to antibiotics are a major concern in the clinic, with almost 15% of the patients presenting with adverse reactions against β-lactam antibiotics. One of the mechanisms involved in this outcome is the modification of proteins by covalent binding of the drug (haptenation). Hence, the interest in identifying the corresponding serum and cellular protein targets. Importantly, haptenation susceptibility and extent can be modulated by the context, including factors affecting protein conformation or the occurrence of other posttranslational modifications. We previously identified the glycolytic enzyme α-enolase as a target for haptenation by amoxicillin, both in cells and in the extracellular milieu. Here, we performed an in vitro study to analyze amoxicillin haptenation of α-enolase using gel-based and activity assays. Moreover, the possible interplay or interference of amoxicillin haptenation with acetylation was also studied in 1D- and 2D-gels that showed decreased haptenation and displacement of the haptenation signal to lower pI spots upon chemical acetylation, respectively. Mass spectrometry identified lysine 239 as the amoxicillin target residue on α enolase, thus suggesting a selective haptenation under our conditions. The amoxicillin binding site and the surrounding interactions were investigated using the α-enolase crystal structure and molecular docking. Altogether, the results obtained provide the basis for the design of novel diagnostic tools or approaches in the study of amoxicillin-induced allergic reactions.

2022-02-17 | PXD029712 | Pride

Amoxicillin Inactivation by Thiol-Catalyzed Cyclization Reduces Protein Haptenation and Antibacterial Potency.

Project description:Serum and cellular proteins are targets for the formation of adducts with the ?-lactam antibiotic amoxicillin. This process could be important for the development of adverse, and in particular, allergic reactions to this antibiotic. In studies exploring protein haptenation by amoxicillin, we observed that reducing agents influenced the extent of amoxicillin-protein adducts formation. Consequently, we show that several thiol-containing compounds, including dithiothreitol, N-acetyl-L-cysteine, and glutathione, perform a nucleophilic attack on the amoxicillin molecule that is followed by an internal rearrangement leading to amoxicillin diketopiperazine, a known amoxicillin metabolite with residual activity. Increased diketopiperazine conversion is also observed with human serum albumin but not with L-cysteine, which mainly forms the amoxicilloyl amide. The effect of thiols is catalytic and can render complete amoxicillin conversion. Interestingly, this process is dependent on the presence of an amino group in the antibiotic lateral chain, as in amoxicillin and ampicillin. Furthermore, it does not occur for other ?-lactam antibiotics, including cefaclor or benzylpenicillin. Biological consequences of thiol-mediated amoxicillin transformation are exemplified by a reduced bacteriostatic action and a lower capacity of thiol-treated amoxicillin to form protein adducts. Finally, modulation of the intracellular redox status through inhibition of glutathione synthesis influenced the extent of amoxicillin adduct formation with cellular proteins. These results open novel perspectives for the understanding of amoxicillin metabolism and actions, including the formation of adducts involved in allergic reactions.

| S-EPMC7065267 | biostudies-literature

Stabilization of Glucocerebrosidase by Active Site Occupancy.

Project description:Glucocerebrosidase (GBA) is a lysosomal β-glucosidase that degrades glucosylceramide. Its deficiency results in Gaucher disease (GD). We examined the effects of active site occupancy of GBA on its structural stability. For this, we made use of cyclophellitol-derived activity-based probes (ABPs) that bind irreversibly to the catalytic nucleophile (E340), and for comparison, we used the potent reversible inhibitor isofagomine. We demonstrate that cyclophellitol ABPs improve the stability of GBA in vitro, as revealed by thermodynamic measurements (Tm increase by 21 °C), and introduce resistance to tryptic digestion. The stabilizing effect of cell-permeable cyclophellitol ABPs is also observed in intact cultured cells containing wild-type GBA, N370S GBA (labile in lysosomes), and L444P GBA (exhibits impaired ER folding): all show marked increases in lysosomal forms of GBA molecules upon exposure to ABPs. The same stabilization effect is observed for endogenous GBA in the liver of wild-type mice injected with cyclophellitol ABPs. Stabilization effects similar to those observed with ABPs were also noted at high concentrations of the reversible inhibitor isofagomine. In conclusion, we provide evidence that the increase in cellular levels of GBA by ABPs and by the reversible inhibitor is in part caused by their ability to stabilize GBA folding, which increases the resistance of GBA against breakdown by lysosomal proteases. These effects are more pronounced in the case of the amphiphilic ABPs, presumably due to their high lipophilic potential, which may promote further structural compactness of GBA through hydrophobic interactions. Our study provides further rationale for the design of chaperones for GBA to ameliorate Gaucher disease.

| S-EPMC5525105 | biostudies-literature

Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli.

Project description:Absolute metabolite concentrations are critical to a quantitative understanding of cellular metabolism, as concentrations impact both the free energies and rates of metabolic reactions. Here we use LC-MS/MS to quantify more than 100 metabolite concentrations in aerobic, exponentially growing Escherichia coli with glucose, glycerol or acetate as the carbon source. The total observed intracellular metabolite pool was approximately 300 mM. A small number of metabolites dominate the metabolome on a molar basis, with glutamate being the most abundant. Metabolite concentration exceeds K(m) for most substrate-enzyme pairs. An exception is lower glycolysis, where concentrations of intermediates are near the K(m) of their consuming enzymes and all reactions are near equilibrium. This may facilitate efficient flux reversibility given thermodynamic and osmotic constraints. The data and analyses presented here highlight the ability to identify organizing metabolic principles from systems-level absolute metabolite concentration data.

| S-EPMC2754216 | biostudies-literature

Substrate-to-Product Conversion Facilitates Active Site Loop Opening in Yeast Enolase: A Molecular Dynamics Study.

Project description:Yeast enolase serves as a prototype for metalloenzymes with labile, catalytic active site metal ions and is important for glycolysis and fermentation processes. Herein, microsecond molecular dynamics simulations of the protein-substrate and protein-product complexes are conducted to elucidate the mechanism of the opening of catalytically important active site loops. These simulations indicate that conversion of substrate to product is accompanied by diminished metal coordination and hydrogen-bonding interactions, as well as enhanced loop flexibility. Moreover, free energy simulations show that the loop opening is endergonic when substrate is bound but exergonic when product is bound. Thus, the conversion to product weakens the association of the loop with the ligand and binding site, thereby facilitating the loop opening after catalysis and enabling product release. These insights about active site loop motions in enzyme catalysis may be useful in guiding enzyme design efforts.

| S-EPMC6919658 | biostudies-literature

Threshold occupancy and specific cation binding modes in the hammerhead ribozyme active site are required for active conformation.

Project description:The relationship between formation of active in-line attack conformations and monovalent (Na(+)) and divalent (Mg(2+)) metal ion binding in hammerhead ribozyme (HHR) has been explored with molecular dynamics simulations. To stabilize repulsions between negatively charged groups, different requirements of the threshold occupancy of metal ions were observed in the reactant and activated precursor states both in the presence and in the absence of a Mg(2+) in the active site. Specific bridging coordination patterns of the ions are correlated with the formation of active in-line attack conformations and can be accommodated in both cases. Furthermore, simulation results suggest that the HHR folds to form an electronegative recruiting pocket that attracts high local concentrations of positive charge. The present simulations help to reconcile experiments that probe the metal ion sensitivity of HHR catalysis and support the supposition that Mg(2+), in addition to stabilizing active conformations, plays a specific chemical role in catalysis.

| S-EPMC2715853 | biostudies-literature

Self-acetylation at the active site of phosphoenolpyruvate carboxykinase (PCK1) controls enzyme activity.

Project description:Acetylation is known to regulate the activity of cytosolic phosphoenolpyruvate carboxykinase (PCK1), a key enzyme in gluconeogenesis, by promoting the reverse reaction of the enzyme (converting phosphoenolpyruvate to oxaloacetate). It is also known that the histone acetyltransferase p300 can induce PCK1 acetylation in cells, but whether that is a direct or indirect function was not known. Here we initially set out to determine whether p300 can acetylate directly PCK1 in vitro. We report that p300 weakly acetylates PCK1, but surprisingly, using several techniques including protein crystallization, mass spectrometry, isothermal titration calorimetry, saturation-transfer difference nuclear magnetic resonance and molecular docking, we found that PCK1 is also able to acetylate itself using acetyl-CoA independently of p300. This reaction yielded an acetylated recombinant PCK1 with a 3-fold decrease in kcat without changes in Km for all substrates. Acetylation stoichiometry was determined for 14 residues, including residues lining the active site. Structural and kinetic analyses determined that site-directed acetylation of K244, located inside the active site, altered this site and rendered the enzyme inactive. In addition, we found that acetyl-CoA binding to the active site is specific and metal dependent. Our findings provide direct evidence for acetyl-CoA binding and chemical reaction with the active site of PCK1 and suggest a newly discovered regulatory mechanism of PCK1 during metabolic stress.

| S-EPMC7948413 | biostudies-literature

Active Site Metal Occupancy and Cyclic Di-GMP Phosphodiesterase Activity of Thermotoga maritima HD-GYP.

Project description:HD-GYPs make up a subclass of the metal-dependent HD phosphohydrolase superfamily and catalyze conversion of cyclic di(3',5')-guanosine monophosphate (c-di-GMP) to 5'-phosphoguanylyl-(3'?5')-guanosine (pGpG) and GMP. Until now, the only reported crystal structure of an HD-GYP that also exhibits c-di-GMP phosphodiesterase activity contains a His/carboxylate ligated triiron active site. However, other structural and phylogenetic correlations indicate that some HD-GYPs contain dimetal active sites. Here we provide evidence that an HD-GYP c-di-GMP phosphodiesterase, TM0186, from Thermotoga maritima can accommodate both di- and trimetal active sites. We show that an as-isolated iron-containing TM0186 has an oxo/carboxylato-bridged diferric site, and that the reduced (diferrous) form is necessary and sufficient to catalyze conversion of c-di-GMP to pGpG, but that conversion of pGpG to GMP requires more than two metals per active site. Similar c-di-GMP phosphodiesterase activities were obtained with divalent iron or manganese. On the basis of activity correlations with several putative metal ligand residue variants and molecular dynamics simulations, we propose that TM0186 can accommodate both di- and trimetal active sites. Our results also suggest that a Glu residue conserved in a subset of HD-GYPs is required for formation of the trimetal site and can also serve as a labile ligand to the dimetal site. Given the anaerobic growth requirement of T. maritima, we suggest that this HD-GYP can function in vivo with either divalent iron or manganese occupying di- and trimetal sites.

| S-EPMC5055813 | biostudies-literature

Human SIRT1 Multispecificity Is Modulated by Active-Site Vicinity Substitutions during Natural Evolution.

Project description:Many enzymes that catalyze protein post-translational modifications can specifically modify multiple target proteins. However, little is known regarding the molecular basis and evolution of multispecificity in these enzymes. Here, we used a combined bioinformatics and experimental approaches to investigate the evolution of multispecificity in the sirtuin-1 (SIRT1) deacetylase. Guided by bioinformatics analysis of SIRT1 orthologs and substrates, we identified and examined important amino acid substitutions that have occurred during the evolution of sirtuins in Metazoa and Fungi. We found that mutation of human SIRT1 at these positions, based on sirtuin orthologs from Fungi, could alter its substrate specificity. These substitutions lead to reduced activity toward K382 acetylated p53 protein, which is only present in Metazoa, without affecting the high activity toward the conserved histone substrates. Results from ancestral sequence reconstruction are consistent with a model in which ancestral sirtuin proteins exhibited multispecificity, suggesting that the multispecificity of some metazoan sirtuins, such as hSIRT1, could be a relatively ancient trait.

| S-EPMC7826192 | biostudies-literature

Acetylation of GATA-1 is required for chromatin occupancy.

Project description:All 3 hematopoietic GATA transcription factors, GATA-1, GATA-2, and GATA-3, are acetylated, although the in vivo role of this modification remains unclear. We examined the functions of an acetylation-defective mutant of GATA-1 in maturing erythroid cells. We found that removal of the acetylation sites in GATA-1 does not impair its nuclear localization, steady-state protein levels, or its ability to bind naked GATA elements in vitro. However, chromatin immunoprecipitation (ChIP) experiments revealed that mutant GATA-1 was dramatically impaired in binding to all examined cellular target sites in vivo, including genes that are normally activated and repressed by GATA-1. Together, these results suggest that acetylation regulates chromatin occupancy of GATA-1. These findings point to a novel function for transcription factor acetylation, perhaps by facilitating protein interactions required for stable association with chromatin templates in vivo.

| S-EPMC1895476 | biostudies-literature

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