RESUMEN

The asymmetric distribution of phospholipids in membranes is a fundamental principle of cellular compartmentalization and organization. Phosphatidylethanolamine (PE), a nonbilayer phospholipid that contributes to organelle shape and function, is synthesized at several subcellular localizations via semiredundant pathways. Previously, we demonstrated in budding yeast that the PE synthase Psd1, which primarily operates on the mitochondrial inner membrane, is additionally targeted to the ER. While ER-localized Psd1 is required to support cellular growth in the absence of redundant pathways, its physiological function is unclear. We now demonstrate that ER-localized Psd1 sublocalizes on the ER to lipid droplet (LD) attachment sites and show it is specifically required for normal LD formation. We also find that the role of phosphatidylserine decarboxylase (PSD) enzymes in LD formation is conserved in other organisms. Thus we have identified PSD enzymes as novel regulators of LDs and demonstrate that both mitochondria and LDs in yeast are organized and shaped by the spatial positioning of a single PE synthesis enzyme.

Asunto(s)

Carboxiliasas/metabolismo , Gotas Lipídicas/metabolismo , Proteínas Mitocondriales/metabolismo , Fosfatidiletanolaminas/metabolismo , Carboxiliasas/fisiología , Retículo Endoplásmico/metabolismo , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/fisiología , Fosfolípidos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo

RESUMEN

Pathogenic and commensal bacteria often have to resist the harsh acidity of the host stomach. The inducible lysine decarboxylase LdcI buffers the cytosol and the local extracellular environment to ensure enterobacterial survival at low pH. Here, we investigate the acid stress-response regulation of Escherichia coli LdcI by combining biochemical and biophysical characterization with negative stain and cryoelectron microscopy (cryo-EM) and wide-field and superresolution fluorescence imaging. Due to deleterious effects of fluorescent protein fusions on native LdcI decamers, we opt for three-dimensional localization of nanobody-labeled endogenous wild-type LdcI in acid-stressed E. coli cells and show that it organizes into distinct patches at the cell periphery. Consistent with recent hypotheses that in vivo clustering of metabolic enzymes often reflects their polymerization as a means of stimulus-induced regulation, we show that LdcI assembles into filaments in vitro at physiologically relevant low pH. We solve the structures of these filaments and of the LdcI decamer formed at neutral pH by cryo-EM and reveal the molecular determinants of LdcI polymerization, confirmed by mutational analysis. Finally, we propose a model for LdcI function inside the enterobacterial cell, providing a structural and mechanistic basis for further investigation of the role of its supramolecular organization in the acid stress response.

Asunto(s)

Carboxiliasas/metabolismo , Microscopía Fluorescente/métodos , Estrés Fisiológico/fisiología , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos/genética , Carboxiliasas/fisiología , Microscopía por Crioelectrón/métodos , Cristalografía por Rayos X/métodos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Concentración de Iones de Hidrógeno , Modelos Moleculares , Unión Proteica/genética , Multimerización de Proteína/genética

RESUMEN

BACKGROUND AND AIMS: Itaconate, a metabolite of the tricarboxylic acid cycle, plays anti-inflammatory roles in macrophages during endotoxemia. The mechanisms underlying its anti-inflammatory roles have been shown to be mediated by the modulation of oxidative stress, an important mechanism of hepatic ischemia-reperfusion (I/R) injury. However, the role of itaconate in liver I/R injury is unknown. APPROACH AND RESULTS: We found that deletion of immune-responsive gene 1 (IRG1), encoding for the enzyme producing itaconate, exacerbated liver injury and systemic inflammation. Furthermore, bone marrow adoptive transfer experiments indicated that deletion of IRG1 in both hematopoietic and nonhematopoietic compartments contributes to the protection mediated by IRG1 after I/R. Interestingly, the expression of IRG1 was up-regulated in hepatocytes after I/R and hypoxia/reoxygenation-induced oxidative stress. Modulation of the IRG1 expression levels in hepatocytes regulated hepatocyte cell death. Importantly, addition of 4-octyl itaconate significantly improved liver injury and hepatocyte cell death after I/R. Furthermore, our data indicated that nuclear factor erythroid 2-related factor 2 (Nrf2) is required for the protective effect of IRG1 on mouse and human hepatocytes against oxidative stress-induced injury. Our studies document the important role of IRG1 in the acute setting of sterile injury induced by I/R. Specifically, we provide evidence that the IRG1/itaconate pathway activates Nrf2-mediated antioxidative response in hepatocytes to protect liver from I/R injury. CONCLUSIONS: Our data expand on the importance of IRG1/itaconate in nonimmune cells and identify itaconate as a potential therapeutic strategy for this unfavorable postsurgical complication.

Asunto(s)

Antiinflamatorios/farmacología , Carboxiliasas/fisiología , Hepatocitos/metabolismo , Hígado/irrigación sanguínea , Factor 2 Relacionado con NF-E2/fisiología , Daño por Reperfusión/prevención & control , Succinatos/farmacología , Animales , Humanos , Hidroliasas/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Estrés Oxidativo , Transducción de Señal/fisiología , Succinatos/uso terapéutico

RESUMEN

Exercise capacity is a strong predictor of all-cause mortality. Skeletal muscle mitochondrial respiratory capacity, its biggest contributor, adapts robustly to changes in energy demands induced by contractile activity. While transcriptional regulation of mitochondrial enzymes has been extensively studied, there is limited information on how mitochondrial membrane lipids are regulated. Here, we show that exercise training or muscle disuse alters mitochondrial membrane phospholipids including phosphatidylethanolamine (PE). Addition of PE promoted, whereas removal of PE diminished, mitochondrial respiratory capacity. Unexpectedly, skeletal muscle-specific inhibition of mitochondria-autonomous synthesis of PE caused respiratory failure because of metabolic insults in the diaphragm muscle. While mitochondrial PE deficiency coincided with increased oxidative stress, neutralization of the latter did not rescue lethality. These findings highlight the previously underappreciated role of mitochondrial membrane phospholipids in dynamically controlling skeletal muscle energetics and function.

Asunto(s)

Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Músculo Esquelético/fisiología , Consumo de Oxígeno , Fosfatidiletanolaminas/metabolismo , Condicionamiento Físico Animal , Animales , Carboxiliasas/fisiología , Tolerancia al Ejercicio , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/patología , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Contracción Muscular , Mioblastos/citología , Mioblastos/metabolismo , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

Low temperature severely influences potato production as the cultivated potato (Solanum tuberosum) is frost sensitive, however the mechanism underlying the freezing tolerance of the potato is largely unknown. In the present research, we studied the transcriptome and metabolome of the freezing-tolerant wild species Solanum acaule (Aca) and freezing-sensitive cultivated S. tuberosum (Tub) to identify the main pathways and important factors related to freezing tolerance. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation indicated that polyamine and amino acid metabolic pathways were specifically upregulated in Aca under cold treatment. The transcriptome changes detected in Aca were accompanied by the specific accumulation of putrescine, saccharides, amino acids and other metabolites. The combination of transcriptome and metabolome analyses revealed that putrescine exhibited an accumulative pattern in accordance with the expression of the arginine decarboxylase gene ADC1. The primary role of putrescine was further confirmed by analyzing all three polyamines (putrescine, spermidine, and spermine) and the genes encoding the corresponding enzymes in two sets of potato genotypes with distinct freezing tolerance, implying that only putrescine and ADC1 were uniquely enhanced by cold in the freezing-tolerant genotypes. The function of putrescine was further analyzed by its exogenous application and the overexpression of SaADC1 in S. tuberosum cv. E3, indicating its important role(s) in cold-acclimated freezing tolerance, which was accompanied with the activation of C-repeat binding factor genes (CBFs). The present research has identified that the ADC1-associated putrescine pathway plays an important role in cold-acclimated freezing tolerance of potato, probably by enhancing the expression of CBF genes.

Asunto(s)

Carboxiliasas/fisiología , Genes de Plantas/fisiología , Proteínas de Plantas/fisiología , Putrescina/metabolismo , Solanum tuberosum/metabolismo , Aclimatación/genética , Carboxiliasas/genética , Respuesta al Choque por Frío , Congelación , Perfilación de la Expresión Génica , Genes de Plantas/genética , Redes y Vías Metabólicas/genética , Metaboloma , Proteínas de Plantas/genética , Solanum tuberosum/fisiología

RESUMEN

γ-Resorcylate decarboxylase (γ-RSD) has evolved to catalyze the reversible decarboxylation of 2,6-dihydroxybenzoate to resorcinol in a nonoxidative fashion. This enzyme is of significant interest because of its potential for the production of γ-resorcylate and other benzoic acid derivatives under environmentally sustainable conditions. Kinetic constants for the decarboxylation of 2,6-dihydroxybenzoate catalyzed by γ-RSD from Polaromonas sp. JS666 are reported, and the enzyme is shown to be active with 2,3-dihydroxybenzoate, 2,4,6-trihydroxybenzoate, and 2,6-dihydroxy-4-methylbenzoate. The three-dimensional structure of γ-RSD with the inhibitor 2-nitroresorcinol (2-NR) bound in the active site is reported. 2-NR is directly ligated to a Mn2+ bound in the active site, and the nitro substituent of the inhibitor is tilted significantly from the plane of the phenyl ring. The inhibitor exhibits a binding mode different from that of the substrate bound in the previously determined structure of γ-RSD from Rhizobium sp. MTP-10005. On the basis of the crystal structure of the enzyme from Polaromonas sp. JS666, complementary density functional calculations were performed to investigate the reaction mechanism. In the proposed reaction mechanism, γ-RSD binds 2,6-dihydroxybenzoate by direct coordination of the active site manganese ion to the carboxylate anion of the substrate and one of the adjacent phenolic oxygens. The enzyme subsequently catalyzes the transfer of a proton to C1 of γ-resorcylate prior to the actual decarboxylation step. The reaction mechanism proposed previously, based on the structure of γ-RSD from Rhizobium sp. MTP-10005, is shown to be associated with high energies and thus less likely to be correct.

Asunto(s)

Carboxiliasas/química , Sitios de Unión , Carboxiliasas/fisiología , Catálisis , Cristalografía por Rayos X , Descarboxilación/fisiología , Hidroxibenzoatos/metabolismo , Cinética , Elementos Estructurales de las Proteínas/fisiología , Resorcinoles/química , Especificidad por Sustrato

RESUMEN

Agmatine (1-amino-4-guanidinobutane), a precursor for polyamine biosynthesis, has been identified as an important neuromodulator with anticonvulsant, antineurotoxic and antidepressant actions in the brain. In this context it has emerged as an important mediator of addiction/satiety pathways associated with alcohol misuse. Consequently, the regulation of the activity of key enzymes in agmatine metabolism is an attractive strategy to combat alcoholism and related addiction disorders. Agmatine results from the decarboxylation of L-arginine in a reaction catalyzed by arginine decarboxylase (ADC), and can be converted to either guanidine butyraldehyde by diamine oxidase (DAO) or putrescine and urea by the enzyme agmatinase (AGM) or the more recently identified AGM-like protein (ALP). In rat brain, agmatine, AGM and ALP are predominantly localised in areas associated with roles in appetitive and craving (drug-reinstatement) behaviors. Thus, inhibitors of AGM or ALP are promising agents for the treatment of addictions. In this review, the properties of DAO, AGM and ALP are discussed with a view to their role in the agmatine metabolism in mammals.

Asunto(s)

Agmatina/metabolismo , Neurotransmisores/metabolismo , Amina Oxidasa (conteniendo Cobre)/fisiología , Animales , Carboxiliasas/fisiología , Humanos , Ureohidrolasas/fisiología

RESUMEN

The mevalonate pathway produces isopentenyl diphosphate (IPP), a building block for polyisoprenoid synthesis, and is a crucial pathway for growth of the human bacterial pathogen Enterococcus faecalis The final enzyme in this pathway, mevalonate diphosphate decarboxylase (MDD), acts on mevalonate diphosphate (MVAPP) to produce IPP while consuming ATP. This essential enzyme has been suggested as a therapeutic target for the treatment of drug-resistant bacterial infections. Here, we report functional and structural studies on the mevalonate diphosphate decarboxylase from E. faecalis (MDDEF). The MDDEF crystal structure in complex with ATP (MDDEF-ATP) revealed that the phosphate-binding loop (amino acids 97-105) is not involved in ATP binding and that the phosphate tail of ATP in this structure is in an outward-facing position pointing away from the active site. This suggested that binding of MDDEF to MVAPP is necessary to guide ATP into a catalytically favorable position. Enzymology experiments show that the MDDEF performs a sequential ordered bi-substrate reaction with MVAPP as the first substrate, consistent with the isothermal titration calorimetry (ITC) experiments. On the basis of ITC results, we propose that this initial prerequisite binding of MVAPP enhances ATP binding. In summary, our findings reveal a substrate-induced substrate-binding event that occurs during the MDDEF-catalyzed reaction. The disengagement of the phosphate-binding loop concomitant with the alternative ATP-binding configuration may provide the structural basis for antimicrobial design against these pathogenic enterococci.

Asunto(s)

Carboxiliasas/metabolismo , Ácido Mevalónico/análogos & derivados , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Sitios de Unión , Carboxiliasas/fisiología , Cristalografía por Rayos X/métodos , Enterococcus faecalis/enzimología , Enterococcus faecalis/metabolismo , Hemiterpenos/biosíntesis , Cinética , Ácido Mevalónico/metabolismo , Compuestos Organofosforados , Especificidad por Sustrato

RESUMEN

Lysine decarboxylase (CadA) converts L-lysine into cadaverine (1,5-pentanediamine), which is an important platform chemical with many industrial applications. Although there have been many efforts to produce cadaverine through the soluble CadA enzyme or Escherichia coli whole cells overexpressing the CadA enzyme, there have been few reports concerning the immobilization of the CadA enzyme. Here, we have prepared a cross-linked enzyme aggregate (CLEA) of E. coli CadA and performed bioconversion using CadACLEA. CadAfree and CadACLEA were characterized for their enzymatic properties. The optimum temperatures of CadAfree and CadACLEA were 60°C and 55°C, respectively. The thermostability of CadACLEA was significantly higher than that of CadAfree. The optimum pH of both enzymes was 6.0. CadAfree could not be recovered after use, whereas CadACLEA was rapidly recovered and the residual activity was 53% after the 10th recycle. These results demonstrate that CadACLEA can be used as a potential catalyst for efficient production of cadaverine.

Asunto(s)

Cadaverina/aislamiento & purificación , Cadaverina/metabolismo , Carboxiliasas/metabolismo , Enzimas Inmovilizadas/metabolismo , Escherichia coli/enzimología , Lisina/metabolismo , Biotransformación , Cadaverina/química , Carboxiliasas/genética , Carboxiliasas/fisiología , Catálisis , Estabilidad de Enzimas , Enzimas Inmovilizadas/química , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Genes Bacterianos , Concentración de Iones de Hidrógeno , Ingeniería Metabólica , Temperatura

RESUMEN

The lactate racemase enzyme (LarA) of Lactobacillus plantarum harbors a (SCS)Ni(II) pincer complex derived from nicotinic acid. Synthesis of the enzyme-bound cofactor requires LarB, LarC, and LarE, which are widely distributed in microorganisms. The functions of the accessory proteins are unknown, but the LarB C terminus resembles aminoimidazole ribonucleotide carboxylase/mutase, LarC binds Ni and could act in Ni delivery or storage, and LarE is a putative ATP-using enzyme of the pyrophosphatase-loop superfamily. Here, we show that LarB carboxylates the pyridinium ring of nicotinic acid adenine dinucleotide (NaAD) and cleaves the phosphoanhydride bond to release AMP. The resulting biscarboxylic acid intermediate is transformed into a bisthiocarboxylic acid species by two single-turnover reactions in which sacrificial desulfurization of LarE converts its conserved Cys176 into dehydroalanine. Our results identify a previously unidentified metabolic pathway from NaAD using unprecedented carboxylase and sulfur transferase reactions to form the organic component of the (SCS)Ni(II) pincer cofactor of LarA. In species where larA is absent, this pathway could be used to generate a pincer complex in other enzymes.

Asunto(s)

Lactobacillus plantarum/enzimología , NAD/análogos & derivados , Níquel/metabolismo , Racemasas y Epimerasas/fisiología , Azufre/metabolismo , Biocatálisis , Carboxiliasas/fisiología , Redes y Vías Metabólicas , NAD/metabolismo

RESUMEN

Soya bean (Glycine max) and grass pea (Lathyrus sativus) seeds are important sources of dietary proteins; however, they also contain antinutritional metabolite oxalic acid (OA). Excess dietary intake of OA leads to nephrolithiasis due to the formation of calcium oxalate crystals in kidneys. Besides, OA is also a known precursor of ß-N-oxalyl-L-α,ß-diaminopropionic acid (ß-ODAP), a neurotoxin found in grass pea. Here, we report the reduction in OA level in soya bean (up to 73%) and grass pea (up to 75%) seeds by constitutive and/or seed-specific expression of an oxalate-degrading enzyme, oxalate decarboxylase (FvOXDC) of Flammulina velutipes. In addition, ß-ODAP level of grass pea seeds was also reduced up to 73%. Reduced OA content was interrelated with the associated increase in seeds micronutrients such as calcium, iron and zinc. Moreover, constitutive expression of FvOXDC led to improved tolerance to the fungal pathogen Sclerotinia sclerotiorum that requires OA during host colonization. Importantly, FvOXDC-expressing soya bean and grass pea plants were similar to the wild type with respect to the morphology and photosynthetic rates, and seed protein pool remained unaltered as revealed by the comparative proteomic analysis. Taken together, these results demonstrated improved seed quality and tolerance to the fungal pathogen in two important legume crops, by the expression of an oxalate-degrading enzyme.

Asunto(s)

Carboxiliasas/genética , Resistencia a la Enfermedad/genética , Glycine max/genética , Lathyrus/genética , Valor Nutritivo/genética , Ácido Oxálico/metabolismo , Carboxiliasas/metabolismo , Carboxiliasas/fisiología , Flammulina/genética , Lathyrus/química , Lathyrus/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Semillas/química , Semillas/metabolismo , Glycine max/química , Glycine max/metabolismo

RESUMEN

Photosynthetic conversion of CO2 to chemicals using cyanobacteria is an attractive approach for direct recycling of CO2 to useful products. 3-Hydroxypropionic acid (3 HP) is a valuable chemical for the synthesis of polymers and serves as a precursor to many other chemicals such as acrylic acid. 3 HP is naturally produced through glycerol metabolism. However, cyanobacteria do not possess pathways for synthesizing glycerol and converting glycerol to 3 HP. Furthermore, the latter pathway requires coenzyme B12, or an oxygen sensitive, coenzyme B12-independent enzyme. These characteristics present major challenges for production of 3 HP using cyanobacteria. To overcome such difficulties, we constructed two alternative pathways in Synechococcus elongatus PCC 7942: a malonyl-CoA dependent pathway and a ß-alanine dependent pathway. Expression of the malonyl-CoA dependent pathway genes (malonyl-CoA reductase and malonate semialdehyde reductase) enabled S. elongatus to synthesize 3 HP to a final titer of 665 mg/L. ß-Alanine dependent pathway expressing S. elongatus produced 3H P to final titer of 186 mg/L. These results demonstrated the feasibility of converting CO2 into 3 HP using cyanobacteria.

Asunto(s)

Dióxido de Carbono/metabolismo , Ácido Láctico/análogos & derivados , Ingeniería Metabólica , Fotosíntesis , Synechococcus/metabolismo , Carboxiliasas/fisiología , Ácido Láctico/biosíntesis , Synechococcus/genética , beta-Alanina/metabolismo

RESUMEN

This manuscript concerns the tissue-specific transcription of mouse and cattle glutamate decarboxylase-like protein 1 (GADL1) and the biochemical activities of human GADL1 recombinant protein. Bioinformatic analysis suggested that GADL1 appears late in evolution, only being found in reptiles, birds, and mammals. RT-PCR determined that GADL1 mRNA is transcribed at high levels in mouse and cattle skeletal muscles and also in mouse kidneys. Substrate screening determined that GADL1, unlike its name implies, has no detectable GAD activity, but it is able to efficiently catalyze decarboxylation of aspartate, cysteine sulfinic acid, and cysteic acid to ß-alanine, hypotaurine, and taurine, respectively. Western blot analysis verified the presence of GADL1 in mouse muscles, kidneys, C2C12 myoblasts, and C2C12 myotubes. Incubation of the supernatant of fresh muscle or kidney extracts with cysteine sulfinic acid resulted in the detection of hypotaurine or taurine in the reaction mixtures, suggesting the possible involvement of GADL1 in taurine biosynthesis. However, when the tissue samples were incubated with aspartate, no ß-alanine production was observed. We proposed several possibilities that might explain the inactivation of ADC activity of GADL1 in tissue protein extracts. Although ß-alanine-producing activity was not detected in the supernatant of tissue protein extracts, its potential role in ß-alanine synthesis cannot be excluded. There are several inhibitors of the ADC activity of GADL1 identified. The discovery of GADL1 biochemical activities, in conjunction with its expression and activities in muscles and kidneys, provides some tangible insight toward establishing its physiological function(s).

Asunto(s)

Carboxiliasas/fisiología , Glutamato Descarboxilasa/metabolismo , Taurina/biosíntesis , Animales , Carboxiliasas/genética , Carboxiliasas/metabolismo , Línea Celular , Ácido Cisteico/metabolismo , Cisteína/análogos & derivados , Cisteína/metabolismo , Riñón/metabolismo , Cinética , Ratones , Modelos Biológicos , Músculos/metabolismo , Mioblastos/metabolismo , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Taurina/análogos & derivados , Taurina/metabolismo , Distribución Tisular , beta-Alanina/metabolismo

RESUMEN

Wybutosine and its derivatives are found in position 37 of tRNA encoding Phe in eukaryotes and archaea. They are believed to play a key role in the decoding function of the ribosome. The second step in the biosynthesis of wybutosine is catalyzed by TYW1 protein, which is a member of the well established class of metalloenzymes called "Radical-SAM." These enzymes use a [4Fe-4S] cluster, chelated by three cysteines in a CX(3)CX(2)C motif, and S-adenosyl-L-methionine (SAM) to generate a 5'-deoxyadenosyl radical that initiates various chemically challenging reactions. Sequence analysis of TYW1 proteins revealed, in the N-terminal half of the enzyme beside the Radical-SAM cysteine triad, an additional highly conserved cysteine motif. In this study we show by combining analytical and spectroscopic methods including UV-visible absorption, Mössbauer, EPR, and HYSCORE spectroscopies that these additional cysteines are involved in the coordination of a second [4Fe-4S] cluster displaying a free coordination site that interacts with pyruvate, the second substrate of the reaction. The presence of two distinct iron-sulfur clusters on TYW1 is reminiscent of MiaB, another tRNA-modifying metalloenzyme whose active form was shown to bind two iron-sulfur clusters. A possible role for the second [4Fe-4S] cluster in the enzyme activity is discussed.

Asunto(s)

Proteínas Arqueales/química , Proteínas Arqueales/genética , Carboxiliasas/fisiología , Oxidorreductasas/fisiología , Pyrococcus abyssi/enzimología , Proteínas de Saccharomyces cerevisiae/fisiología , Secuencia de Aminoácidos , Proteínas Arqueales/fisiología , Carboxiliasas/genética , Catálisis , Cromatografía Líquida de Alta Presión , Clonación Molecular , Análisis por Conglomerados , Cisteína/genética , Espectroscopía de Resonancia por Spin del Electrón , Guanosina/análogos & derivados , Guanosina/química , Proteínas Hierro-Azufre/química , Espectrometría de Masas/métodos , Modelos Químicos , Datos de Secuencia Molecular , Oxidorreductasas/genética , Pyrococcus abyssi/genética , Ácido Pirúvico/química , ARN de Transferencia/metabolismo , S-Adenosilmetionina/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Rayos Ultravioleta

RESUMEN

BACKGROUND: Dental biofilms contain a protein that inhibits mammalian cell growth, possibly lysine decarboxylase from Eikenella corrodens. This enzyme decarboxylates lysine, an essential amino acid for dentally attached cell turnover in gingival sulci. Lysine depletion may stop this turnover, impairing the barrier to bacterial compounds. The aims of this study are to determine biofilm lysine and cadaverine contents before oral hygiene restriction (OHR) and their association with plaque index (PI) and gingival crevicular fluid (GCF) after OHR for 1 week. METHODS: Laser-induced fluorescence after capillary electrophoresis was used to determine lysine and cadaverine contents in dental biofilm, tongue biofilm, and saliva before OHR and in dental biofilm after OHR. RESULTS: Before OHR, lysine and cadaverine contents of dental biofilm were similar and 10-fold greater than in saliva or tongue biofilm. After 1 week of OHR, the biofilm content of cadaverine increased and that of lysine decreased, consistent with greater biofilm lysine decarboxylase activity. Regression indicated that PI and GCF exudation were positively related to biofilm lysine after OHR, unless biofilm lysine exceeded the minimal blood plasma content, in which case PI was further increased but GCF exudation was reduced. CONCLUSIONS: After OHR, lysine decarboxylase activity seems to determine biofilm lysine content and biofilm accumulation. When biofilm lysine exceeds minimal blood plasma content after OHR, less GCF appeared despite more biofilm. Lysine appears important for biofilm accumulation and the epithelial barrier to bacterial proinflammatory agents. Inhibiting lysine decarboxylase may retard the increased GCF exudation required for microbial development and gingivitis.

Asunto(s)

Biopelículas/crecimiento & desarrollo , Carboxiliasas/fisiología , Gingivitis/etiología , Lisina/análisis , Adulto , Cadaverina/análisis , Índice de Placa Dental , Electroforesis Capilar , Femenino , Estudios de Seguimiento , Líquido del Surco Gingival/química , Líquido del Surco Gingival/metabolismo , Humanos , Lisina/sangre , Masculino , Higiene Bucal , Putrescina/análisis , Saliva/química , Adulto Joven

RESUMEN

The proton-pumping NADH:ubiquinone oxidoreductase, respiratory complex I, couples the electron transfer from NADH to ubiquinone with the translocation of protons across the membrane. In Escherichia coli the complex is made up of 13 different subunits encoded by the so-called nuo-genes. Mutants, in which each of the nuo-genes was individually disrupted by the insertion of a resistance cartridge were unable to assemble a functional complex I. Each disruption resulted in the loss of complex I-mediated activity and the failure to extract a structurally intact complex. Thus, all nuo-genes are required either for the assembly or the stability of a functional E. coli complex I. The three subunits comprising the soluble NADH dehydrogenase fragment of the complex were detected in the cytoplasm of several nuo-mutants as one distinct band after BN-PAGE. It is discussed that the fully assembled NADH dehydrogenase fragment represents an assembly intermediate of the E. coli complex I. A partially assembled complex I bound to the membrane was detected in the nuoK and nuoL mutants, respectively. Overproduction of the ΔNuoL variant resulted in the accumulation of two populations of a partially assembled complex in the cytoplasmic membranes. Both populations are devoid of NuoL. One population is enzymatically active, while the other is not. The inactive population is missing cluster N2 and is tightly associated with the inducible lysine decarboxylase. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.

Asunto(s)

Complejo I de Transporte de Electrón/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Carboxiliasas/metabolismo , Carboxiliasas/fisiología , Membrana Celular/enzimología , Membrana Celular/metabolismo , Citoplasma/enzimología , Espectroscopía de Resonancia por Spin del Electrón , Complejo I de Transporte de Electrón/antagonistas & inhibidores , Complejo I de Transporte de Electrón/metabolismo , Escherichia coli/enzimología , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiología , Furanos/farmacología , Eliminación de Gen , Expresión Génica , Proteínas Hierro-Azufre/metabolismo , Multimerización de Proteína , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo

RESUMEN

Enteroaggregative Escherichia coli (EAEC) strains are important diarrheal pathogens. EAEC strains are defined by their characteristic stacked-brick pattern of adherence to epithelial cells but show heterogeneous virulence and have different combinations of adhesin and toxin genes. Pathoadaptive deletions in the lysine decarboxylase (cad) genes have been noted among hypervirulent E. coli subtypes of Shigella and enterohemorrhagic E. coli. To test the hypothesis that cad deletions might account for heterogeneity in EAEC virulence, we developed a Caenorhabditis elegans pathogenesis model. Well-characterized EAEC strains were shown to colonize and kill C. elegans, and differences in virulence could be measured quantitatively. Of 49 EAEC strains screened for lysine decarboxylase activity, 3 tested negative. Most notable is isolate 101-1, which was recovered in Japan, from the largest documented EAEC outbreak. EAEC strain 101-1 was unable to decarboxylate lysine in vitro due to deletions in cadA and cadC, which, respectively, encode lysine decarboxylase and a transcriptional activator of the cadAB genes. Strain 101-1 was significantly more lethal to C. elegans than control strain OP50. Lethality was attenuated when the lysine decarboxylase defect was complemented from a multicopy plasmid and in single copy. In addition, restoring lysine decarboxylase function produced derivatives of 101-1 deficient in aggregative adherence to cultured human epithelial cells. Lysine decarboxylase inactivation is pathoadapative in an important EAEC outbreak strain, and deletion of cad genes could produce hypervirulent EAEC lineages in the future. These results suggest that loss, as well as gain, of genetic material can account for heterogeneous virulence among EAEC strains.

Asunto(s)

Caenorhabditis elegans/microbiología , Carboxiliasas/fisiología , Escherichia coli/patogenicidad , Animales , Biopelículas , Carboxiliasas/genética , Adhesión Celular , Células Cultivadas , Brotes de Enfermedades , Eliminación de Gen , Humanos , Virulencia

RESUMEN

The mechanism of wild-type and R37A mutant Pseudomonas dacunhae aspartate beta-decarboxylase (ABDC) was studied by rapid-scanning stopped-flow spectrophotometry. Mixing wild-type ABDC with 50 mM disodium l-Asp resulted in the formation of a 325 nm absorption peak within the dead time of the stopped-flow instrument, likely the ketimine of pyridoxamine 5'-phosphate and oxaloacetate or pyruvate. After consumption of the l-Asp, the 360 nm feature of the resting enzyme was restored. Thus, the 325 nm species is a catalytically competent intermediate. In contrast, mixing wild-type ABDC with the disodium salt of either threo- or erythro-beta-hydroxy-dl-Asp at 50 mM resulted in a much slower formation of the 325 nm complex, with an apparent rate constant of approximately 1 or 0.006 s(-1), respectively. When wild-type ABDC is mixed with disodium succinate, a nonreactive analogue of l-Asp, formation of a new peak at 425 nm is observed. The apparent rate constant for formation of the 425 nm band exhibits a hyperbolic dependence on succinate concentration, showing that there is a rapid binding equilibrium, followed by a slower reaction in which the internal aldimine is protonated on the Schiff base N. Hydrostatic pressure shifts the spectrum from the 425 nm form to the 360 nm form, consistent with a conformational change. It is likely that the binding of substrate or analogues induces a conformational change that releases strain in the Lys pyridoxal 5'-phosphate Schiff base and increases the pK(a), resulting in protonation of the Schiff base to initiate transaldimination. Mixing of R37A mutant ABDC with 50 mM l-Asp also results in the formation of the 325 nm complex, but with an apparent rate constant of 0.2 s(-1), at least 5000-fold slower than the rate of wild-type ABDC. In contrast to wild-type ABDC, R37A ABDC shows no change in the cofactor spectrum when mixed with disodium succinate. These results suggest that Arg-37, a conserved active site residue in ABDC, plays a role in modulating the pK(a) of the pyridoxal 5'-phosphate complexes during catalysis.

Asunto(s)

Proteínas Bacterianas/química , Carboxiliasas/química , Pseudomonas/enzimología , Ácido Aspártico/análogos & derivados , Ácido Aspártico/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Carboxiliasas/genética , Carboxiliasas/fisiología , Presión Hidrostática , Cinética , Mutación , Conformación Proteica , Fosfato de Piridoxal/química , Espectrofotometría , Estereoisomerismo , Ácido Succínico/química

RESUMEN

The UDP-sugar interconverting enzymes involved in UDP-GlcA metabolism are well described in eukaryotes but less is known in prokaryotes. Here we identify and characterize a gene (RsU4kpxs) from Ralstonia solanacearum str. GMI1000, which encodes a dual function enzyme not previously described. One activity is to decarboxylate UDP-glucuronic acid to UDP-beta-l-threo-pentopyranosyl-4''-ulose in the presence of NAD(+). The second activity converts UDP-beta-l-threo-pentopyranosyl-4''-ulose and NADH to UDP-xylose and NAD(+), albeit at a lower rate. Our data also suggest that following decarboxylation, there is stereospecific protonation at the C5 pro-R position. The identification of the R. solanacearum enzyme enables us to propose that the ancestral enzyme of UDP-xylose synthase and UDP-apiose/UDP-xylose synthase was diverged to two distinct enzymatic activities in early bacteria. This separation gave rise to the current UDP-xylose synthase in animal, fungus, and plant as well as to the plant Uaxs and bacterial ArnA and U4kpxs homologs.

Asunto(s)

Oxidorreductasas de Alcohol/química , Carboxiliasas/química , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Complejos Multienzimáticos/química , Plantas/microbiología , Ralstonia solanacearum/metabolismo , Azúcares de Uridina Difosfato/química , Uridina Difosfato Xilosa/química , Oxidorreductasas de Alcohol/fisiología , Secuencia de Aminoácidos , Carboxiliasas/fisiología , Clonación Molecular , Escherichia coli/metabolismo , Espectroscopía de Resonancia Magnética , Modelos Biológicos , Modelos Químicos , Datos de Secuencia Molecular , Complejos Multienzimáticos/fisiología , Filogenia , Homología de Secuencia de Aminoácido

RESUMEN

Unlike most other organisms, the essential five-step coenzyme A biosynthetic pathway has not been fully resolved in yeast. Specifically, the genes encoding the phosphopantothenoylcysteine decarboxylase (PPCDC) activity still remain unidentified. Sequence homology analyses suggest three candidates-Ykl088w, Hal3 and Vhs3-as putative PPCDC enzymes in Saccharomyces cerevisiae. Notably, Hal3 and Vhs3 have been characterized as negative regulatory subunits of the Ppz1 protein phosphatase. Here we show that YKL088w does not encode a third Ppz1 regulatory subunit, and that the essential roles of Ykl088w and the Hal3 and Vhs3 pair are complementary, cannot be interchanged and can be attributed to PPCDC-related functions. We demonstrate that while known eukaryotic PPCDCs are homotrimers, the active yeast enzyme is a heterotrimer that consists of Ykl088w and Hal3/Vhs3 monomers that separately provides two essential catalytic residues. Our results unveil Hal3 and Vhs3 as moonlighting proteins involved in both CoA biosynthesis and protein phosphatase regulation.

Asunto(s)

Carboxiliasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Coenzima A/biosíntesis , Fosfoproteínas Fosfatasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencia de Bases , Carboxiliasas/genética , Carboxiliasas/fisiología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiología , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/fisiología , Unión Proteica , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiología