RESUMEN
Terpenes are high-value chemicals which can be produced by engineered cyanobacteria from sustainable resources, solar energy, water and CO2. We previously reported that the euryhaline unicellular cyanobacteria Synechocystis sp. PCC 6803 (S.6803) and Synechococcus sp. PCC 7002 (S.7002) produce farnesene and limonene, respectively, more efficiently than other terpenes. In the present study, we attempted to enhance farnesene production in S.6803 and limonene production in S.7002. Practically, we tested the influence of key cyanobacterial enzymes acting in carbon fixation (RubisCO, PRK, CcmK3 and CcmK4), utilization (CrtE, CrtR and CruF) and storage (PhaA and PhaB) on terpene production in S.6803, and we compared some of the findings with the data obtained in S.7002. We report that the overproduction of RubisCO from S.7002 and PRK from Cyanothece sp. PCC 7425 increased farnesene production in S.6803, but not limonene production in S.7002. The overexpression of the crtE genes (synthesis of terpene precursors) from S.6803 or S.7002 did not increase farnesene production in S.6803. In contrast, the overexpression of the crtE gene from S.6803, but not S.7002, increased farnesene production in S.7002, emphasizing the physiological difference between these two model cyanobacteria. Furthermore, the deletion of the crtR and cruF genes (carotenoid synthesis) and phaAB genes (carbon storage) did not increase the production of farnesene in S.6803. Finally, as a containment strategy of genetically modified strains of S.6803, we report that the deletion of the ccmK3K4 genes (carboxysome for CO2 fixation) did not affect the production of limonene, but decreased the production of farnesene in S.6803.
Asunto(s)
Sesquiterpenos , Synechococcus , Synechocystis , Limoneno , Synechococcus/genética , Synechocystis/genética , Dióxido de Carbono , Ribulosa-Bifosfato Carboxilasa , Terpenos , Ciclo del CarbonoAsunto(s)
Antígenos Bacterianos/inmunología , Glucolípidos/inmunología , Mycobacterium tuberculosis/inmunología , Vacunas contra la Tuberculosis/inmunología , Tuberculosis/prevención & control , Antígenos Bacterianos/química , Glucolípidos/química , Humanos , Mycobacterium tuberculosis/química , Trehalosa , Tuberculosis/inmunología , Tuberculosis/microbiología , Vacunas contra la Tuberculosis/químicaRESUMEN
The presentation of lipid antigens to T cells is mediated by the CD1 proteins. Purified functional CD1/lipid complexes are valuable tools to investigate such immune processes. Here, we describe how these complexes can be prepared in vitro, how they can be purified by chromatofocusing and how to control their antigen-loading status by isoelectric focusing.
Asunto(s)
Antígenos CD1/aislamiento & purificación , Focalización Isoeléctrica/métodos , Antígenos CD1/química , Antígenos CD1/metabolismo , Humanos , Estructura Terciaria de ProteínaRESUMEN
CD1b-restricted T lymphocytes recognize a large diversity of mycobacterial lipids, which differ in their hydrophilic heads and the structure of their acyl appendages. Both moieties participate in the antigenicity of lipid Ags, but the structural constraints governing binding to CD1b and generation of antigenic CD1b:lipid Ag complexes are still poorly understood. Here, we investigated the structural requirements conferring antigenicity to Mycobacterium tuberculosis sulfoglycolipid Ags using a combination of CD1b:lipid binding and T cell activation assays with both living dendritic cells and plate-bound recombinant soluble CD1b. Comparison of the antigenicity of a panel of synthetic analogs, sharing the same trehalose-sulfate polar head, but differing in the structure of their acyl tails, shows that the number of C-methyl substituents on the fatty acid, the configuration of the chiral centers, and the respective localization of the two different acyl chains on the sugar moiety govern TCR recognition and T lymphocyte activation. These studies have major implications for the design of sulfoglycolipid analogs with potential use as tuberculosis subunit vaccines.
Asunto(s)
Antígenos CD1/metabolismo , Glucolípidos/inmunología , Mycobacterium tuberculosis/inmunología , Linfocitos T/inmunología , Animales , Antígenos CD1/inmunología , Células Dendríticas , Ácidos Grasos/química , Glucolípidos/química , Glucolípidos/metabolismo , Humanos , Activación de Linfocitos , Ratones , Estructura Molecular , Mycobacterium tuberculosis/química , Unión Proteica , Vacunas contra la TuberculosisRESUMEN
Complexes between CD1 molecules and self or microbial glycolipids represent important immunogenic ligands for specific subsets of T cells. However, the function of one of the CD1 family members, CD1e, has yet to be determined. Here, we show that the mycobacterial antigens hexamannosylated phosphatidyl-myo-inositols (PIM6) stimulate CD1b-restricted T cells only after partial digestion of the oligomannose moiety by lysosomal alpha-mannosidase and that soluble CD1e is required for this processing. Furthermore, recombinant CD1e was able to bind glycolipids and assist in the digestion of PIM6. We propose that, through this form of glycolipid editing, CD1e helps expand the repertoire of glycolipidic T cell antigens to optimize antimicrobial immune responses.
Asunto(s)
Presentación de Antígeno , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/metabolismo , Antígenos CD1/metabolismo , Glucolípidos/inmunología , Fosfatidilinositoles/inmunología , Fosfatidilinositoles/metabolismo , Acilación , Células Presentadoras de Antígenos/inmunología , Antígenos CD1/química , Antígenos CD1/genética , Antígenos CD1/inmunología , Línea Celular Tumoral , Células Dendríticas/enzimología , Células Dendríticas/inmunología , Glucolípidos/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Activación de Linfocitos , Modelos Moleculares , Mycobacterium tuberculosis/inmunología , Conformación Proteica , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Solubilidad , Linfocitos T/inmunología , Transfección , alfa-Manosidasa/inmunologíaRESUMEN
Dihydroxyacetone (Dha) kinases are homologous proteins that use different phosphoryl donors, a multiphosphoryl protein of the phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system in bacteria, ATP in animals, plants, and some bacteria. The Dha kinase of Escherichia coli consists of three subunits, DhaK and DhaL, which are colinear to the ATP-dependent Dha kinases of eukaryotes, and the multiphosphoryl protein DhaM. Here we show the crystal structure of the DhaK subunit in complex with Dha at 1.75 A resolution. DhaK is a homodimer with a fold consisting of two six-stranded mixed beta-sheets surrounded by nine alpha-helices and a beta-ribbon covering the exposed edge strand of one sheet. The core of the N-terminal domain has an alpha/beta fold common to subunits of carbohydrate transporters and transcription regulators of the phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system. The core of the C-terminal domain has a fold similar to the C-terminal domain of the cell-division protein FtsZ. A molecule of Dha is covalently bound in hemiaminal linkage to the N epsilon 2 of His-230. The hemiaminal does not participate in covalent catalysis but is the chemical basis for discrimination between short-chain carbonyl compounds and polyols. Paralogs of Dha kinases occur in association with transcription regulators of the TetR/QacR and the SorC families, pointing to their biological role as sensors in signaling.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Secuencia de Aminoácidos , Bacterias/enzimología , Proteínas Bacterianas/química , Cristalografía por Rayos X , Proteínas de Escherichia coli/química , Modelos Moleculares , Datos de Secuencia Molecular , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Enzyme I (EI), the first component of the phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS), consists of an N-terminal domain with the phosphorylation site (His-189) and a C-terminal domain with the PEP binding site. Here we use C3-substituted PEP analogues as substrates and inhibitors and the EI(C502A) mutant to characterize structure-activity relationships of the PEP binding site. EI(C502A) is 10 000 times less active than wild-type EI [EI(wt)] with PEP as the substrate, whereas the two forms are equally active with ZClPEP. Cys-502 acts as an acid-base catalyst which stereospecifically protonates the pyruvoyl enolate at C3. The electron-withdrawing chlorine of ZClPEP can compensate for the lack of Cys-502, and in this case, the released 3-Cl-enolate is protonated nonstereospecifically. Several PEP analogues were assayed as inhibitors and as substrates. The respective K(I)/K(m) ratios vary between 3 and 40 for EI(wt), but they are constant and around unity for EI(C502A). EI(wt) with PEP as the substrate is inhibited by oxalate, whereas EI(C502A) with ZClPEP is not. The different behavior of EI(wt) and EI(C502A) toward the PEP analogues and oxalate suggests that the PEP binding site of EI(wt) exists in a "closed" and an "open" form. The open to closed transition is triggered by the interaction of the substrate with Cys-502. The closed conformation is sterically disfavored by C3-modified substrate analogues such as ZClPEP and ZMePEP. If site closure does not occur as with EI(C502A) and bulky substrates, the transition state is stabilized by electron dispersion to the electron-withdrawing substituent at C3.
Asunto(s)
Cisteína/química , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/química , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/metabolismo , Fosfotransferasas (Aceptor del Grupo Nitrogenado)/química , Fosfotransferasas (Aceptor del Grupo Nitrogenado)/metabolismo , Sitios de Unión , Catálisis , Cisteína/fisiología , Dimerización , Inhibidores Enzimáticos/metabolismo , Isomerismo , Cinética , Mutación , Oxalatos/metabolismo , Fosfoenolpiruvato/análogos & derivados , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/genética , Fosfotransferasas (Aceptor del Grupo Nitrogenado)/genética , Conformación Proteica , Protones , Relación Estructura-ActividadRESUMEN
Thirteen glucose analogues bearing electrophilic groups were synthesized (five of them for the first time) and screened as inhibitors of the glucose transporter (EIIGlc) of the Escherichia coli phosphoenolpyruvate-sugar phosphotransferase system (PTS). 2',3'-Epoxypropyl beta-d-glucopyranoside (3a) is an inhibitor and also a pseudosubstrate. Five analogues are inhibitors of nonvectorial Glc phosphorylation by EIIGlc but not pseudosubstrates. They are selective for EIIGlc as demonstrated by comparison with EIIMan, another Glc-specific but structurally different transporter. 3a is the only analogue that inhibits EIIGlc by binding to the high-affinity cytoplasmic binding site and also strongly inhibits sugar uptake mediated by this transporter. The most potent inhibitor in vitro, methyl 6,7-anhydro-d,l-glycero-alpha-d-gluco-heptopyranoside (1d), preferentially interacts with the low-affinity cytoplasmic site but only weakly inhibits Glc uptake. Binding and/or phosphorylation from the cytoplasmic side of EIIGlc is more permissive than sugar binding and/or translocation of substrates via the periplasmic site. EIIGlc is rapidly inactivated by the 6-O-bromoacetyl esters of methyl alpha-d-glucopyranoside (1a) and methyl alpha-d-mannopyranoside (1c), methyl 6-deoxy-6-isothiocyanato-alpha-d-glucopyranoside (1e), beta-d-glucopyranosyl isothiocyanate (3c) and beta-d-glucopyranosyl phenyl isothiocyanate (3d). Phosphorylation of EIIGlc protects, indicating that inactivation occurs by alkylation of Cys421. Glc does not protect, but sensitizes EIIGlc for inactivation by 1e and 3d, which is interpreted as the effect of glucose-induced conformational changes in the dimeric transporter. Glc also sensitizes EIIGlc for inactivation by 1a and 1c of uptake by starved cells. This indicates that Cys421 which is located on the cytoplasmic domain of EIIGlc becomes transiently accessible to substrate analogues on the periplasmic side of the transporter.
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Compuestos Epoxi/química , Compuestos Epoxi/farmacología , Proteínas de Escherichia coli/antagonistas & inhibidores , Glucósidos/química , Glucósidos/farmacología , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/antagonistas & inhibidores , Sitios de Unión , Bioquímica/métodos , Transporte Biológico , Cisteína/química , Cisteína/metabolismo , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/síntesis química , Compuestos Epoxi/síntesis química , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Glucosa/química , Glucosa/metabolismo , Glucósidos/síntesis química , Glucósidos/metabolismo , Pruebas de Sensibilidad Microbiana , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/efectos de los fármacos , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/metabolismo , Fosforilación , Relación Estructura-ActividadRESUMEN
The synthesis of 10 new phosphoenolpyruvate (PEP) analogues with modifications in the phosphate and the carboxylate function is described. Included are two potential irreversible inhibitors of PEP-utilizing enzymes. One incorporates a reactive chloromethylphosphonate function replacing the phosphate group of PEP. The second contains a chloromethyl group substituting for the carboxylate function of PEP. An improved procedure for the preparation of the known (Z)- and (E)-3-chloro-PEP is also given. The isomers were obtained as a 4 : 1 mixture, resolved by anion-exchange chromatography after the last reaction step. The stereochemistry of the two isomers was unequivocally assigned from the (3)J(H-C) coupling constants between the carboxylate carbons and the vinyl protons. All of these and other known PEP-analogues were tested as reversible and irreversible inhibitors of Mg2+- and Mn2+- activated PEP-utilizing enzymes: enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), pyruvate kinase, PEP carboxylase and enolase. Without exception, the most potent inhibitors were those with substitution of a vinyl proton. Modification of the phosphate and the carboxylate groups resulted in less effective compounds. Enzyme I was the least tolerant to such modifications. Among the carboxylate-modified analogues, only those replaced by a negatively charged group inhibited pyruvate kinase and enolase. Remarkably, the activity of PEP carboxylase was stimulated by derivatives with neutral groups at this position in the presence of Mg2+, but not with Mn2+. For the irreversible inhibition of these enzymes, (Z)-3-Cl-PEP was found to be a very fast-acting and efficient suicide inhibitor of enzyme I (t(1/2) = 0.7 min).
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/antagonistas & inhibidores , Fosfoenolpiruvato/química , Fosfoenolpiruvato/farmacología , Bioquímica/métodos , Evaluación Preclínica de Medicamentos , Activación Enzimática , Inhibidores Enzimáticos/metabolismo , Isomerismo , Fosfoenolpiruvato/análogos & derivados , Fosfoenolpiruvato/metabolismo , Fosfoenolpiruvato Carboxilasa/antagonistas & inhibidores , Fosfoenolpiruvato Carboxilasa/metabolismo , Fosfopiruvato Hidratasa/antagonistas & inhibidores , Fosfopiruvato Hidratasa/metabolismo , Fosfotransferasas (Aceptor del Grupo Nitrogenado)/antagonistas & inhibidores , Piruvato Quinasa/antagonistas & inhibidores , Piruvato Quinasa/metabolismo , Relación Estructura-ActividadRESUMEN
Four phosphoenolpyruvate (PEP) derivatives, carrying reactive or activable chemical functions in each of the three chemical regions of PEP, were assayed as alternative substrates of enzyme I (EI) of the Escherichia coli PEP:glucose phosphotransferase system. The Z- and E-isomers of 3-chlorophosphoenolpyruvate (3-Cl-PEP) were substrates, presenting K(m) values of 0.08 and 0.12 mm, respectively, very similar to the K(m) of 0.14 mm measured for PEP, and k(cat) of 40 and 4 min(-1), compared with 2,200 min(-1), for PEP. The low catalytic efficiency of these substrates permits the study of activity at in vivo EI concentrations. Z-Cl-PEP was a competitive inhibitor of PEP with a K(I) of 0.4 mm. E-Cl-PEP was not an inhibitor. Compounds 3 and 4, obtained by modification of the carboxylic and phosphate groups of PEP, were neither substrates nor inhibitors of EI, highlighting the importance of these functionalities for recognition by EI. Z-Cl-PEP is a suicide inhibitor. About 10-50 turnovers sufficed to inactivate EI completely. Such a property can be exploited to reveal and quantitate phosphoryl transfer from EI to other proteins at in vivo concentrations. Inactivation was saturatable in Z-Cl-PEP, with an apparent K(m)(inact) of 0.2-0.4 mm. The rate of inactivation increased with the concentration of EI, indicating a preferential or exclusive reaction with the dimeric form of EI. E-Cl-PEP inactivates EI much more slowly, and unlike PEP, it did not protect against inactivation by Z-Cl-PEP. This and the ineffectiveness of E-Cl-PEP as a competitive inhibitor have been related to the presence of two EI active species. Cys-502 of EI was identified by mass spectrometry as the reacting residue. The C502A EI mutant showed less than 0.06% wild-type activity. Sequence alignments and comparisons of x-ray structures of different PEP-utilizing enzymes indicate that Cys-502 might serve as a proton donor during catalysis.