RESUMEN
An arsenate reductase (Car1) from the Bacteroidetes species Rufibacter tibetensis 1351T was isolated from the Tibetan Plateau. The strain exhibits resistance to arsenite [As(III)] and arsenate [As(V)] and reduces As(V) to As(III). Here we shed light on the mechanism of enzymatic reduction by Car1. AlphaFold2 structure prediction, active site energy minimization, and steady-state kinetics of wild-type and mutant enzymes give insight into the catalytic mechanism. Car1 is structurally related to calcineurin-like metallophosphoesterases (MPPs). It functions as a binuclear metal hydrolase with limited phosphatase activity, particularly relying on the divalent metal Ni2+. As an As(V) reductase, it displays metal promiscuity and is coupled to the thioredoxin redox cycle, requiring the participation of two cysteine residues, Cys74 and Cys76. These findings suggest that Car1 evolved from a common ancestor of extant phosphatases by incorporating a redox function into an existing MPP catalytic site. Its proposed mechanism of arsenate reduction involves Cys74 initiating a nucleophilic attack on arsenate, leading to the formation of a covalent intermediate. Next, a nucleophilic attack of Cys76 leads to the release of As(III) and the formation of a surface-exposed Cys74-Cys76 disulfide, ready for reduction by thioredoxin.
Asunto(s)
Arseniato Reductasas , Bacteroidetes , Dominio Catalítico , Oxidación-Reducción , Arseniato Reductasas/metabolismo , Arseniato Reductasas/genética , Arseniato Reductasas/química , Bacteroidetes/enzimología , Bacteroidetes/genética , Arseniatos/metabolismo , Cinética , Monoéster Fosfórico Hidrolasas/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/química , Catálisis , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Arsenitos/metabolismoRESUMEN
The function of ascorbate peroxidase-related (APX-R) proteins, present in all green photosynthetic eukaryotes, remains unclear. This study focuses on APX-R from Chlamydomonas reinhardtii, namely, ascorbate peroxidase 2 (APX2). We showed that apx2 mutants exhibited a faster oxidation of the photosystem I primary electron donor, P700, upon sudden light increase and a slower re-reduction rate compared to the wild type, pointing to a limitation of plastocyanin. Spectroscopic, proteomic and immunoblot analyses confirmed that the phenotype was a result of lower levels of plastocyanin in the apx2 mutants. The redox state of P700 did not differ between wild type and apx2 mutants when the loss of function in plastocyanin was nutritionally complemented by growing apx2 mutants under copper deficiency. In this case, cytochrome c6 functionally replaces plastocyanin, confirming that lower levels of plastocyanin were the primary defect caused by the absence of APX2. Overall, the results presented here shed light on an unexpected regulation of plastocyanin level under copper-replete conditions, induced by APX2 in Chlamydomonas.
Asunto(s)
Ascorbato Peroxidasas , Chlamydomonas reinhardtii , Mutación , Plastocianina , Plastocianina/metabolismo , Plastocianina/genética , Ascorbato Peroxidasas/metabolismo , Ascorbato Peroxidasas/genética , Chlamydomonas reinhardtii/metabolismo , Chlamydomonas reinhardtii/genética , Cobre/metabolismo , Oxidación-Reducción , Complejo de Proteína del Fotosistema I/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Citocromos c6/metabolismo , Citocromos c6/genética , Proteómica/métodos , LuzRESUMEN
Neutrophils are often the major leukocyte at sites of mycobacterial infection, yet little is known about their ability to kill mycobacteria. In this study we have investigated whether the potent antibacterial oxidant hypochlorous acid (HOCl) contributes to killing of Mycobacterium smegmatis when this bacterium is phagocytosed by human neutrophils. We found that M. smegmatis were ingested by neutrophils into intracellular phagosomes but were killed slowly. We measured a t 1/2 of 30 min for the survival of M. smegmatis inside neutrophils, which is 5 times longer than that reported for Staphylococcus aureus and 15 times longer than Escherichia coli Live-cell imaging indicated that neutrophils generated HOCl in phagosomes containing M. smegmatis; however, inhibition of HOCl production did not alter the rate of bacterial killing. Also, the doses of HOCl that are likely to be produced inside phagosomes failed to kill isolated bacteria. Lethal doses of reagent HOCl caused oxidation of mycothiol, the main low-m.w. thiol in this bacterium. In contrast, phagocytosed M. smegmatis maintained their original level of reduced mycothiol. Collectively, these findings suggest that M. smegmatis can cope with the HOCl that is produced inside neutrophil phagosomes. A mycothiol-deficient mutant was killed by neutrophils at the same rate as wild-type bacteria, indicating that mycothiol itself is not the main driver of M. smegmatis resistance. Understanding how M. smegmatis avoids killing by phagosomal HOCl could provide new opportunities to sensitize pathogenic mycobacteria to destruction by the innate immune system.
Asunto(s)
Antibacterianos/metabolismo , Ácido Hipocloroso/metabolismo , Infecciones por Mycobacterium no Tuberculosas/inmunología , Mycobacterium smegmatis/fisiología , Neutrófilos/metabolismo , Fagosomas/metabolismo , Células Cultivadas , Cisteína/metabolismo , Glicopéptidos/metabolismo , Humanos , Evasión Inmune , Inmunidad Innata , Inositol/metabolismo , Infecciones por Mycobacterium no Tuberculosas/microbiología , Neutrófilos/inmunología , FagocitosisRESUMEN
Pharmacological AMPK activation represents an attractive approach for the treatment of type 2 diabetes (T2D). AMPK activation increases skeletal muscle glucose uptake, but there is controversy as to whether AMPK activation also inhibits hepatic glucose production (HGP) and pharmacological AMPK activators can have off-target effects that contribute to their anti-diabetic properties. The main aim was to investigate the effects of 991 and other direct AMPK activators on HGP and determine whether the observed effects were AMPK-dependent. In incubated hepatocytes, 991 substantially decreased gluconeogenesis from lactate, pyruvate and glycerol, but not from other substrates. Hepatocytes from AMPKß1-/- mice had substantially reduced liver AMPK activity, yet the inhibition of glucose production by 991 persisted. Also, the glucose-lowering effect of 991 was still seen in AMPKß1-/- mice subjected to an intraperitoneal pyruvate tolerance test. The AMPK-independent mechanism by which 991 treatment decreased gluconeogenesis could be explained by inhibition of mitochondrial pyruvate uptake and inhibition of mitochondrial sn-glycerol-3-phosphate dehydrogenase-2. However, 991 and new-generation direct small-molecule AMPK activators antagonized glucagon-induced gluconeogenesis in an AMPK-dependent manner. Our studies support the notion that direct pharmacological activation of hepatic AMPK as well as inhibition of pyruvate uptake could be an option for the treatment of T2D-linked hyperglycemia.
Asunto(s)
Diabetes Mellitus Tipo 2 , Glucagón , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Glucagón/metabolismo , Gluconeogénesis , Glucosa/metabolismo , Ácido Láctico/metabolismo , Hígado/metabolismo , Ratones , Ácido Pirúvico/metabolismoRESUMEN
Despite being initially regarded as a metabolic waste product, lactate is now considered to serve as a primary fuel for the tricarboxylic acid cycle in cancer cells. At the core of lactate metabolism, lactate dehydrogenases (LDHs) catalyze the interconversion of lactate to pyruvate and as such represent promising targets in cancer therapy. However, direct inhibition of the LDH active site is challenging from physicochemical and selectivity standpoints. However, LDHs are obligate tetramers. Thus, targeting the LDH tetrameric interface has emerged as an appealing strategy. In this work, we examine a dimeric construct of truncated human LDH to search for new druggable sites. We report the identification and characterization of a new cluster of interactions in the LDH tetrameric interface. Using nanoscale differential scanning fluorimetry, chemical denaturation, and mass photometry, we identified several residues (E62, D65, L71, and F72) essential for LDH tetrameric stability. Moreover, we report a family of peptide ligands based on this cluster of interactions. We next demonstrated these ligands to destabilize tetrameric LDHs through binding to this new tetrameric interface using nanoscale differential scanning fluorimetry, NMR water-ligand observed via gradient spectroscopy, and microscale thermophoresis. Altogether, this work provides new insights on the LDH tetrameric interface as well as valuable pharmacological tools for the development of LDH tetramer disruptors.
Asunto(s)
Mapeo Epitopo/métodos , L-Lactato Deshidrogenasa/metabolismo , Humanos , L-Lactato Deshidrogenasa/genética , L-Lactato Deshidrogenasa/fisiología , Lactato Deshidrogenasas/metabolismo , Ácido Láctico/metabolismo , Ligandos , Imagen por Resonancia Magnética/métodos , Péptidos/metabolismoRESUMEN
Hydrogen peroxide (H2O2) is an important messenger molecule for diverse cellular processes. H2O2 oxidizes proteinaceous cysteinyl thiols to sulfenic acid, also known as S-sulfenylation, thereby affecting the protein conformation and functionality. Although many proteins have been identified as S-sulfenylation targets in plants, site-specific mapping and quantification remain largely unexplored. By means of a peptide-centric chemoproteomics approach, we mapped 1,537 S-sulfenylated sites on more than 1,000 proteins in Arabidopsis thaliana cells. Proteins involved in RNA homeostasis and metabolism were identified as hotspots for S-sulfenylation. Moreover, S-sulfenylation frequently occurred on cysteines located at catalytic sites of enzymes or on cysteines involved in metal binding, hinting at a direct mode of action for redox regulation. Comparison of human and Arabidopsis S-sulfenylation datasets provided 155 conserved S-sulfenylated cysteines, including Cys181 of the Arabidopsis MITOGEN-ACTIVATED PROTEIN KINASE4 (AtMAPK4) that corresponds to Cys161 in the human MAPK1, which has been identified previously as being S-sulfenylated. We show that, by replacing Cys181 of recombinant AtMAPK4 by a redox-insensitive serine residue, the kinase activity decreased, indicating the importance of this noncatalytic cysteine for the kinase mechanism. Altogether, we quantitatively mapped the S-sulfenylated cysteines in Arabidopsis cells under H2O2 stress and thereby generated a comprehensive view on the S-sulfenylation landscape that will facilitate downstream plant redox studies.
Asunto(s)
Arabidopsis/metabolismo , Proteínas/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Dominio Catalítico/fisiología , Cisteína/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Oxidación-Reducción , ARN/metabolismo , Serina/metabolismo , Transducción de Señal/fisiología , Ácidos Sulfénicos/metabolismoRESUMEN
Corynebacterium diphtheriae is a human pathogen that causes diphtheria. In response to immune system-induced oxidative stress, C. diphtheriae expresses antioxidant enzymes, among which are methionine sulfoxide reductase (Msr) enzymes, which are critical for bacterial survival in the face of oxidative stress. Although some aspects of the catalytic mechanism of the Msr enzymes have been reported, several details still await full elucidation. Here, we solved the solution structure of C. diphtheriae MsrB (Cd-MsrB) and unraveled its catalytic and oxidation-protection mechanisms. Cd-MsrB catalyzes methionine sulfoxide reduction involving three redox-active cysteines. Using NMR heteronuclear single-quantum coherence spectra, kinetics, biochemical assays, and MS analyses, we show that the conserved nucleophilic residue Cys-122 is S-sulfenylated after substrate reduction, which is then resolved by a conserved cysteine, Cys-66, or by the nonconserved residue Cys-127. We noted that the overall structural changes during the disulfide cascade expose the Cys-122-Cys-66 disulfide to recycling through thioredoxin. In the presence of hydrogen peroxide, Cd-MsrB formed reversible intra- and intermolecular disulfides without losing its Cys-coordinated Zn2+, and only the nonconserved Cys-127 reacted with the low-molecular-weight (LMW) thiol mycothiol, protecting it from overoxidation. In summary, our structure-function analyses reveal critical details of the Cd-MsrB catalytic mechanism, including a major structural rearrangement that primes the Cys-122-Cys-66 disulfide for thioredoxin reduction and a reversible protection against excessive oxidation of the catalytic cysteines in Cd-MsrB through intra- and intermolecular disulfide formation and S-mycothiolation.
Asunto(s)
Biocatálisis , Corynebacterium diphtheriae/enzimología , Disulfuros/metabolismo , Metionina Sulfóxido Reductasas/metabolismo , Safrol/análogos & derivados , Dominio Catalítico , Secuencia Conservada , Cisteína/metabolismo , Glicopéptidos/metabolismo , Inositol/metabolismo , Espectroscopía de Resonancia Magnética , Metionina Sulfóxido Reductasas/química , Modelos Moleculares , Oxidación-Reducción , Safrol/metabolismo , Especificidad por Sustrato , Ácidos Sulfénicos/metabolismo , Reductasa de Tiorredoxina-Disulfuro/metabolismo , Tiorredoxinas/metabolismo , Zinc/metabolismoRESUMEN
Electrostatic forces are important for protein folding and are favored targets of protein engineering. However, interactions between charged residues are difficult to study because of the complex network of interactions found in most proteins. We have designed a purposely simple system to investigate this problem by systematically introducing individual and pairs of charged and titratable residues in a protein otherwise free of such residues. We used constant pH molecular dynamics simulations, NMR spectroscopy, and thermodynamic double mutant cycles to probe the structure and energetics of the interaction between the charged residues. We found that the partial burial of surface charges contributes to a shift in pKa value, causing an aspartate to titrate in the neutral pH range. Additionally, the interaction between pairs of residues was found to be highly context dependent, with some pairs having no apparent preferential interaction, while other pairs would engage in coupled titration forming a highly stabilized salt bridge. We find good agreement between experiments and simulations and use the simulations to rationalize our observations and to provide a detailed mechanistic understanding of the electrostatic interactions.
Asunto(s)
Celulasa/química , Electricidad Estática , Ácido Aspártico/química , Celulasa/genética , Cellulomonas/enzimología , Histidina/química , Simulación de Dinámica Molecular , Mutación , Conformación Proteica , Dominios Proteicos/genética , Desplegamiento Proteico , TermodinámicaRESUMEN
Heat stress induces misfolding and aggregation of proteins unless they are guarded by chaperone systems. Here, we examined the function of the glutaredoxin GRXS17, a member of thiol reductase families in the model plant Arabidopsis (Arabidopsis thaliana). GRXS17 is a nucleocytosolic monothiol glutaredoxin consisting of an N-terminal thioredoxin domain and three CGFS active-site motif-containing GRX domains that coordinate three iron-sulfur (Fe-S) clusters in a glutathione-dependent manner. As an Fe-S cluster-charged holoenzyme, GRXS17 is likely involved in the maturation of cytosolic and nuclear Fe-S proteins. In addition to its role in cluster biogenesis, GRXS17 presented both foldase and redox-dependent holdase activities. Oxidative stress in combination with heat stress induced loss of its Fe-S clusters followed by subsequent formation of disulfide bonds between conserved active-site cysteines in the corresponding thioredoxin domains. This oxidation led to a shift of GRXS17 to a high-molecular-weight complex and thus activated its holdase activity in vitro. Moreover, GRXS17 was specifically involved in plant tolerance to moderate high temperature and protected root meristematic cells from heat-induced cell death. Finally, GRXS17 interacted with a different set of proteins upon heat stress, possibly protecting them from heat injuries. Therefore, we propose that the Fe-S cluster enzyme GRXS17 is an essential guard that protects proteins against moderate heat stress, likely through a redox-dependent chaperone activity. We reveal the mechanism of an Fe-S cluster-dependent activity shift that converts the holoenzyme GRXS17 into a holdase, thereby preventing damage caused by heat stress.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Glutarredoxinas/metabolismo , Respuesta al Choque Térmico , Estrés Oxidativo , Termotolerancia , Arabidopsis , Proteínas de Arabidopsis/genética , Glutarredoxinas/genética , PolimerizacionRESUMEN
Hydrogen peroxide (H2O2) is a strong oxidant capable of oxidizing cysteinyl thiolates, yet only a few cysteine-containing proteins have exceptional reactivity toward H2O2 One such example is the prokaryotic transcription factor OxyR, which controls the antioxidant response in bacteria, and which specifically and rapidly reduces H2O2 In this study, we present crystallographic evidence for the H2O2-sensing mechanism and H2O2-dependent structural transition of Corynebacterium glutamicum OxyR by capturing the reduced and H2O2-bound structures of a serine mutant of the peroxidatic cysteine, and the full-length crystal structure of disulfide-bonded oxidized OxyR. In the H2O2-bound structure, we pinpoint the key residues for the peroxidatic reduction of H2O2, and relate this to mutational assays showing that the conserved active-site residues T107 and R278 are critical for effective H2O2 reduction. Furthermore, we propose an allosteric mode of structural change, whereby a localized conformational change arising from H2O2-induced intramolecular disulfide formation drives a structural shift at the dimerization interface of OxyR, leading to overall changes in quaternary structure and an altered DNA-binding topology and affinity at the catalase promoter region. This study provides molecular insights into the overall OxyR transcription mechanism regulated by H2O2.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Corynebacterium glutamicum/metabolismo , Peróxido de Hidrógeno/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Sitios de Unión/genética , Catalasa/química , Catalasa/genética , Catalasa/metabolismo , Corynebacterium glutamicum/genética , Cristalografía por Rayos X , Genes Bacterianos , Cinética , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Estructura Cuaternaria de Proteína , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
Protein cysteine thiols are post-translationally modified under oxidative stress conditions. Illuminated chloroplasts are one of the important sources of hydrogen peroxide (H2 O2 ) and are highly sensitive to environmental stimuli, yet a comprehensive view of the oxidation-sensitive chloroplast proteome is still missing. By targeting the sulfenic acid YAP1C-trapping technology to the plastids of light-grown Arabidopsis cells, we identified 132 putatively sulfenylated plastid proteins upon H2 O2 pulse treatment. Almost half of the sulfenylated proteins are enzymes of the amino acid metabolism. Using metabolomics, we observed a reversible decrease in the levels of the amino acids Ala, Asn, Cys, Gln, Glu, His, Ile, Leu, Lys, Phe, Ser, Thr and Val after H2 O2 treatment, which is in line with an anticipated decrease in the levels of the glycolysis and tricarboxylic acid metabolites. Through the identification of an organelle-tailored proteome, we demonstrated that the subcellular targeting of the YAP1C probe enables us to study in vivo cysteine sulfenylation at the organellar level. All in all, the identification of these oxidation events in plastids revealed that several enzymes of the amino acid metabolism rapidly undergo cysteine oxidation upon oxidative stress.
Asunto(s)
Aminoácidos/metabolismo , Cisteína/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plastidios/metabolismo , Oxidación-Reducción , Estrés Oxidativo , Proteínas de Plantas/genética , Ácidos Sulfénicos/metabolismoRESUMEN
Thioredoxins (Trxs) are antioxidant proteins that are conserved among all species. These proteins have been extensively studied and perform reducing reactions on a broad range of substrates. Here, we identified Caulobacter crescentus Trx1 (CCNA_03653; CcTrx1) as an oxidoreductase that is involved in the cell cycle progression of this model bacterium and is required to sustain life. Intriguingly, the abundance of CcTrx1 varies throughout the C. crescentus cell cycle: although the expression of CcTrx1 is induced in stalked cells, right before DNA replication initiation, CcTrx1 is actively degraded by the ClpXP protease in predivisional cells. Importantly, we demonstrated that regulation of the abundance of CcTrx1 is crucial for cell growth and survival as modulating CcTrx1 levels leads to cell death. Finally, we also report a comprehensive biochemical and structural characterization of this unique and essential Trx. The requirement to precisely control the abundance of CcTrx1 for cell survival underlines the importance of redox control for optimal cell cycle progression in C. crescentus.
Asunto(s)
Proteínas Bacterianas/metabolismo , Caulobacter crescentus/metabolismo , Ciclo Celular , Regulación Bacteriana de la Expresión Génica , Modelos Moleculares , Oxidorreductasas/metabolismo , Tiorredoxinas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Caulobacter crescentus/citología , Caulobacter crescentus/crecimiento & desarrollo , Secuencia Conservada , Cristalografía por Rayos X , Replicación del ADN , Endopeptidasa Clp/metabolismo , Técnicas de Inactivación de Genes , Viabilidad Microbiana , Oxidorreductasas/antagonistas & inhibidores , Oxidorreductasas/química , Oxidorreductasas/genética , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Proteolisis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Especificidad por Sustrato , Tiorredoxinas/antagonistas & inhibidores , Tiorredoxinas/química , Tiorredoxinas/genéticaRESUMEN
Hydrogen peroxide (H2O2) can act as a signaling molecule that influences various aspects of plant growth and development, including stress signaling and cell death. To analyze molecular mechanisms that regulate the response to increased H2O2 levels in plant cells, we focused on the photorespiration-dependent peroxisomal H2O2 production in Arabidopsis thaliana mutants lacking CATALASE2 (CAT2) activity (cat2-2). By screening for second-site mutations that attenuate the PSII maximum efficiency (Fv'/Fm') decrease and lesion formation linked to the cat2-2 phenotype, we discovered that a mutation in SHORT-ROOT (SHR) rescued the cell death phenotype of cat2-2 plants under photorespiration-promoting conditions. SHR deficiency attenuated H2O2-dependent gene expression, oxidation of the glutathione pool, and ascorbate depletion in a cat2-2 genetic background upon exposure to photorespiratory stress. Decreased glycolate oxidase and catalase activities together with accumulation of glycolate further implied that SHR deficiency impacts the cellular redox homeostasis by limiting peroxisomal H2O2 production. The photorespiratory phenotype of cat2-2 mutants did not depend on the SHR functional interactor SCARECROW and the sugar signaling component ABSCISIC ACID INSENSITIVE4, despite the requirement for exogenous sucrose for cell death attenuation in cat2-2 shr-6 double mutants. Our findings reveal a link between SHR and photorespiratory H2O2 production that has implications for the integration of developmental and stress responses.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Catalasa/metabolismo , Factores de Transcripción/deficiencia , Factores de Transcripción/metabolismo , Arabidopsis/citología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Catalasa/genética , Muerte Celular/genética , Muerte Celular/fisiología , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Peróxido de Hidrógeno/metabolismo , Oxidación-Reducción , Plantas Modificadas Genéticamente/citología , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Transducción de Señal/genética , Transducción de Señal/fisiología , Factores de Transcripción/genéticaRESUMEN
The Obg protein family belongs to the TRAFAC (translation factor) class of P-loop GTPases and is conserved from bacteria to eukaryotes. Essential roles in many different cellular processes have been suggested for the Obg protein from Escherichia coli (ObgE), and we recently showed that it is a central regulator of bacterial persistence. Here, we report the first crystal structure of ObgE at 1.85-Å resolution in the GDP-bound state, showing the characteristic N-terminal domain and a central G domain that are common to all Obg proteins. ObgE also contains an intrinsically disordered C-terminal domain, and we show here that this domain specifically contributed to GTP binding, whereas it did not influence GDP binding or GTP hydrolysis. Biophysical analysis, using small angle X-ray scattering and multi-angle light scattering experiments, revealed that ObgE is a monomer in solution, regardless of the bound nucleotide. In contrast to recent suggestions, our biochemical analyses further indicate that ObgE is neither activated by K+ ions nor by homodimerization. However, the ObgE GTPase activity was stimulated upon binding to the ribosome, confirming the ribosome-dependent GTPase activity of the Obg family. Combined, our data represent an important step toward further unraveling the detailed molecular mechanism of ObgE, which might pave the way to further studies into how this GTPase regulates bacterial physiology, including persistence.
Asunto(s)
Proteínas de Escherichia coli/química , Escherichia coli/química , Proteínas de Unión al GTP Monoméricas/química , Potasio/química , Multimerización de Proteína , Cationes Monovalentes/química , Cationes Monovalentes/metabolismo , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Potasio/metabolismo , Dominios ProteicosRESUMEN
The Mycobacterium tuberculosis rv2466c gene encodes an oxidoreductase enzyme annotated as DsbA. It has a CPWC active-site motif embedded within its thioredoxin fold domain and mediates the activation of the prodrug TP053, a thienopyrimidine derivative that kills both replicating and nonreplicating bacilli. However, its mode of action and actual enzymatic function in M. tuberculosis have remained enigmatic. In this study, we report that Rv2466c is essential for bacterial survival under H2O2 stress. Further, we discovered that Rv2466c lacks oxidase activity; rather, it receives electrons through the mycothiol/mycothione reductase/NADPH pathway to activate TP053, preferentially via a dithiol-disulfide mechanism. We also found that Rv2466c uses a monothiol-disulfide exchange mechanism to reduce S-mycothiolated mixed disulfides and intramolecular disulfides. Genetic, phylogenetic, bioinformatics, structural, and biochemical analyses revealed that Rv2466c is a novel mycothiol-dependent reductase, which represents a mycoredoxin cluster of enzymes within the DsbA family different from the glutaredoxin cluster to which mycoredoxin-1 (Mrx1 or Rv3198A) belongs. To validate this DsbA-mycoredoxin cluster, we also characterized a homologous enzyme of Corynebacterium glutamicum (NCgl2339) and observed that it demycothiolates and reduces a mycothiol arsenate adduct with kinetic properties different from those of Mrx1. In conclusion, our work has uncovered a DsbA-like mycoredoxin that promotes mycobacterial resistance to oxidative stress and reacts with free mycothiol and mycothiolated targets. The characterization of the DsbA-like mycoredoxin cluster reported here now paves the way for correctly classifying similar enzymes from other organisms.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Modelos Moleculares , Mycobacterium tuberculosis/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Profármacos/farmacología , Proteína Disulfuro Isomerasas/metabolismo , Pirimidinas/farmacología , Activación Metabólica , Antibacterianos/química , Antibacterianos/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Biocatálisis , Dominio Catalítico , Cristalografía por Rayos X , Cisteína/metabolismo , Pruebas Antimicrobianas de Difusión por Disco , Drogas en Investigación/química , Drogas en Investigación/metabolismo , Drogas en Investigación/farmacología , Eliminación de Gen , Conformación Molecular , Simulación del Acoplamiento Molecular , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/crecimiento & desarrollo , Oxidación-Reducción , Filogenia , Profármacos/química , Profármacos/metabolismo , Conformación Proteica , Proteína Disulfuro Isomerasas/química , Proteína Disulfuro Isomerasas/genética , Pirimidinas/química , Pirimidinas/metabolismo , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Protein lysine acylations, such as succinylation and acetylation, are important post-translational modification (PTM) mechanisms, with key roles in cellular regulation. Antibody-based affinity enrichment, high-resolution liquid chromatography mass spectrometry analysis, and integrated bioinformatics analysis were used to characterize the lysine succinylome (Ksuc ) and acetylome (Kace ) of rice leaves. In total, 2,593 succinylated and 1,024 acetylated proteins were identified, of which 723 were simultaneously acetylated and succinylated. Proteins involved in photosynthetic carbon metabolism such as the large and small subunits of RuBisCO, ribosomal functions, and other key processes were subject to both PTMs. Preliminary insights into oxidant-induced changes to the rice acetylome and succinylome were gained from treatments with hydrogen peroxide. Exposure to oxidative stress did not regulate global changes in the rice acetylome or succinylome but rather led to modifications on a specific subset of the identified sites. De-succinylation of recombinant catalase (CATA) and glutathione S-transferase (OsGSTU6) altered the activities of these enzymes showing that this PTM may have a regulatory function. These findings not only greatly extend the list of acetylated and/or succinylated proteins but they also demonstrate the close cooperation between these PTMs in leaf proteins with key metabolic functions.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Lisina/metabolismo , Oryza/fisiología , Proteínas de Plantas/metabolismo , Procesamiento Proteico-Postraduccional , Proteoma , Acetilación , Aminoacilación , Cromatografía Liquida , Espectrometría de Masas , Estrés Oxidativo , Fotosíntesis , Hojas de la Planta/fisiología , Proteínas de Plantas/genética , Ácido Succínico/metabolismoRESUMEN
Plant malate dehydrogenase (MDH) isoforms are found in different cell compartments and function in key metabolic pathways. It is well known that the chloroplastic NADP-dependent MDH activities are strictly redox regulated and controlled by light. However, redox dependence of other NAD-dependent MDH isoforms have been less studied. Here, we show by in vitro biochemical characterization that the major cytosolic MDH isoform (cytMDH1) is sensitive to H2O2 through sulfur oxidation of cysteines and methionines. CytMDH1 oxidation affects the kinetics, secondary structure, and thermodynamic stability of cytMDH1. Moreover, MS analyses and comparison of crystal structures between the reduced and H2O2-treated cytMDH1 further show that thioredoxin-reversible homodimerization of cytMDH1 through Cys330 disulfide formation protects the protein from overoxidation. Consistently, we found that cytosolic thioredoxins interact specifically with cytMDH in a yeast two-hybrid system. Importantly, we also show that cytosolic and chloroplastic, but not mitochondrial NAD-MDH activities are sensitive to H2O2 stress in Arabidopsis. NAD-MDH activities decreased both in a catalase2 mutant and in an NADP-thioredoxin reductase mutant, emphasizing the importance of the thioredoxin-reducing system to protect MDH from oxidation in vivo. We propose that the redox switch of the MDH activity contributes to adapt the cell metabolism to environmental constraints.
Asunto(s)
Arabidopsis/metabolismo , Malato Deshidrogenasa/metabolismo , Estrés Oxidativo , Arabidopsis/enzimología , Citosol/metabolismo , Peróxido de Hidrógeno/metabolismo , Oxidación-ReducciónRESUMEN
BACKGROUND: Glutathione transferases play an important role as detoxifying enzymes. In A. thaliana, elevated levels of reactive oxygen species (ROS), provoked during biotic and abiotic stress, influence the activity of GSTU23. The aim of this study is to determine the impact of oxidative stress on the function and structure of GSTU23. METHODS: The impact of oxidation on the function of GSTU23 was studied using a glutathione transferase biochemical assay and mass spectrometry. With kinetics, circular dichroism and thermodynamics, we compared reduced with oxidized GSTU23. X-ray crystal structures of GSTU23 visualize the impact of oxidation on methionines and cysteines. RESULTS: In the presence of 100µM H2O2, oxidation of the methionine side-chain to a sulfoxide is the prominent post-translational modification, which can be reduced by C. diphtheriae MsrA and MsrB. However, increasing the level to 200µM H2O2 results in a reversible intramolecular disulfide between Cys65-Cys110, which is substrate for glutaredoxin. Under these oxidizing conditions, GSTU23 undergoes a structural change and forms a more favourable enzyme-substrate complex to overcome kcat decrease. CONCLUSIONS AND SIGNIFICANCE: At lower H2O2 levels (100µM), GSTU23 forms methionine sulfoxides. Specifically, oxidation of Met14, located near the catalytic Ser13, could interfere with both GSH binding and catalytic activation. At higher H2O2 levels (200µM), the Cys65-Cys110 disulfide bond protects other cysteines and also methionines from overoxidation. This study shows the impact of oxidative stress on GSTU23 regulated by methionine sulfoxide reductases and glutaredoxin, and the mechanisms involved in maintaining its catalytic functionality under oxidizing conditions.
Asunto(s)
Arabidopsis/enzimología , Disulfuros/metabolismo , Glutatión Transferasa/química , Glutatión Transferasa/metabolismo , Estrés Oxidativo , Sustancias Protectoras , Arabidopsis/crecimiento & desarrollo , Catálisis , Glutarredoxinas/metabolismo , Disulfuro de Glutatión/metabolismo , Glutatión Transferasa/genética , Peróxido de Hidrógeno/metabolismo , Metionina/análogos & derivados , Metionina/metabolismo , Sustancias Protectoras/metabolismoRESUMEN
Cysteines are among the rarest amino acids in nature, and are both functionally and structurally very important for proteins. The ability of cysteines to form disulfide bonds is especially relevant, both for constraining the folded state of the protein and for performing enzymatic duties. But how does the variation record of human proteins reflect their functional importance and structural role, especially with regard to deleterious mutations? We created HUMCYS, a manually curated dataset of single amino acid variants that (1) have a known disease/neutral phenotypic outcome and (2) cause the loss of a cysteine, in order to investigate how mutated cysteines relate to structural aspects such as surface accessibility and cysteine oxidation state. We also have developed a sequence-based in silico cysteine oxidation predictor to overcome the scarcity of experimentally derived oxidation annotations, and applied it to extend our analysis to classes of proteins for which the experimental determination of their structure is technically challenging, such as transmembrane proteins. Our investigation shows that we can gain insights into the reason behind the outcome of cysteine losses in otherwise uncharacterized proteins, and we discuss the possible molecular mechanisms leading to deleterious phenotypes, such as the involvement of the mutated cysteine in a structurally or enzymatically relevant disulfide bond.
Asunto(s)
Cisteína/genética , Modelos Biológicos , Mutación , Oxidación-Reducción , Algoritmos , Sustitución de Aminoácidos , Codón , Biología Computacional/métodos , Bases de Datos Genéticas , Estudios de Asociación Genética , Humanos , Espacio Intracelular/metabolismo , Polimorfismo de Nucleótido Simple , Transporte de Proteínas , Reproducibilidad de los Resultados , Programas Informáticos , Navegador WebRESUMEN
Exposure of bacteria to NO results in the nitrosylation of cysteine thiols in proteins and low molecular weight thiols such as GSH. The cells possess enzymatic systems that catalyze the denitrosylation of these modified sulfurs. An important player in these systems is thioredoxin (Trx), a ubiquitous, cytoplasmic oxidoreductase that can denitrosylate proteins in vivo and S-nitrosoglutathione (GSNO) in vitro However, a periplasmic or extracellular denitrosylase has not been identified, raising the question of how extracytoplasmic proteins are repaired after nitrosative damage. In this study, we tested whether DsbG and DsbC, two Trx family proteins that function in reducing pathways in the Escherichia coli periplasm, also possess denitrosylating activity. Both DsbG and DsbC are poorly reactive toward GSNO. Moreover, DsbG is unable to denitrosylate its specific substrate protein, YbiS. Remarkably, by borrowing the CGPC active site of E. coli Trx-1 in combination with a T200M point mutation, we transformed DsbG into an enzyme highly reactive toward GSNO and YbiS. The pKa of the nucleophilic cysteine, as well as the redox and thermodynamic properties of the engineered DsbG are dramatically changed and become similar to those of E. coli Trx-1. X-ray structural insights suggest that this results from a loss of two direct hydrogen bonds to the nucleophilic cysteine sulfur in the DsbG mutant. Our results highlight the plasticity of the Trx structural fold and reveal that the subtle change of the number of hydrogen bonds in the active site of Trx-like proteins is the key factor that thermodynamically controls reactivity toward nitrosylated compounds.