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1.
J Biol Chem ; 298(5): 101903, 2022 05.
Artículo en Inglés | MEDLINE | ID: mdl-35398092

RESUMEN

The sugars streptose and dihydrohydroxystreptose (DHHS) are unique to the bacteria Streptomyces griseus and Coxiella burnetii, respectively. Streptose forms the central moiety of the antibiotic streptomycin, while DHHS is found in the O-antigen of the zoonotic pathogen C. burnetii. Biosynthesis of these sugars has been proposed to follow a similar path to that of TDP-rhamnose, catalyzed by the enzymes RmlA, RmlB, RmlC, and RmlD, but the exact mechanism is unclear. Streptose and DHHS biosynthesis unusually requires a ring contraction step that could be performed by orthologs of RmlC or RmlD. Genome sequencing of S. griseus and C. burnetii has identified StrM and CBU1838 proteins as RmlC orthologs in these respective species. Here, we demonstrate that both enzymes can perform the RmlC 3'',5'' double epimerization activity necessary to support TDP-rhamnose biosynthesis in vivo. This is consistent with the ring contraction step being performed on a double epimerized substrate. We further demonstrate that proton exchange is faster at the 3''-position than the 5''-position, in contrast to a previously studied ortholog. We additionally solved the crystal structures of CBU1838 and StrM in complex with TDP and show that they form an active site highly similar to those of the previously characterized enzymes RmlC, EvaD, and ChmJ. These results support the hypothesis that streptose and DHHS are biosynthesized using the TDP pathway and that an RmlD paralog most likely performs ring contraction following double epimerization. This work will support the elucidation of the full pathways for biosynthesis of these unique sugars.


Asunto(s)
Antígenos Bacterianos/biosíntesis , Carbohidrato Epimerasas , Coxiella burnetii/enzimología , Streptomyces griseus/enzimología , Carbohidrato Epimerasas/genética , Azúcares de Nucleósido Difosfato/biosíntesis , Nucleótidos de Timina/biosíntesis
2.
J Nat Prod ; 86(7): 1677-1689, 2023 07 28.
Artículo en Inglés | MEDLINE | ID: mdl-37327570

RESUMEN

Formicamycins and their biosynthetic intermediates the fasamycins are polyketide antibiotics produced by Streptomyces formicae KY5 from a pathway encoded by the for biosynthetic gene cluster. In this work the ability of Streptomyces coelicolor M1146 and the ability of Saccharopolyspora erythraea Δery to heterologously express the for biosynthetic gene cluster were assessed. This led to the identification of eight new glycosylated fasamycins modified at different phenolic groups with either a monosaccharide (glucose, galactose, or glucuronic acid) or a disaccharide comprised of a proximal hexose (either glucose or galactose), with a terminal pentose (arabinose) moiety. In contrast to the respective aglycones, minimal inhibitory screening assays showed these glycosylated congeners lacked antibacterial activity.


Asunto(s)
Galactosa , Streptomyces coelicolor , Galactosa/metabolismo , Antibacterianos/metabolismo , Streptomyces coelicolor/genética , Familia de Multigenes , Glucosa/metabolismo
4.
Nature ; 544(7648): 65-70, 2017 04 06.
Artículo en Inglés | MEDLINE | ID: mdl-28329766

RESUMEN

The metabolism of carbohydrate polymers drives microbial diversity in the human gut microbiota. It is unclear, however, whether bacterial consortia or single organisms are required to depolymerize highly complex glycans. Here we show that the gut bacterium Bacteroides thetaiotaomicron uses the most structurally complex glycan known: the plant pectic polysaccharide rhamnogalacturonan-II, cleaving all but 1 of its 21 distinct glycosidic linkages. The deconstruction of rhamnogalacturonan-II side chains and backbone are coordinated to overcome steric constraints, and the degradation involves previously undiscovered enzyme families and catalytic activities. The degradation system informs revision of the current structural model of rhamnogalacturonan-II and highlights how individual gut bacteria orchestrate manifold enzymes to metabolize the most challenging glycan in the human diet.


Asunto(s)
Bacteroides thetaiotaomicron/enzimología , Bacteroides thetaiotaomicron/metabolismo , Biocatálisis , Tracto Gastrointestinal/microbiología , Glicósido Hidrolasas/metabolismo , Pectinas/química , Pectinas/metabolismo , Bacteroides thetaiotaomicron/crecimiento & desarrollo , Boratos/química , Boratos/metabolismo , Dominio Catalítico , Microbioma Gastrointestinal , Glicósido Hidrolasas/química , Glicósido Hidrolasas/clasificación , Humanos , Modelos Moleculares , Especificidad por Sustrato
5.
Int J Mol Sci ; 23(6)2022 Mar 10.
Artículo en Inglés | MEDLINE | ID: mdl-35328413

RESUMEN

Xylan is one of the major structural components of the plant cell wall. Xylan present in the human diet reaches the large intestine undigested and becomes a substrate to species of the gut microbiota. Here, we characterised the capacity of Limosilactobacillus reuteri and Blautia producta strains to utilise xylan derivatives. We showed that L. reuteri ATCC 53608 and B. producta ATCC 27340 produced ß-D-xylosidases, enabling growth on xylooligosaccharide (XOS). The recombinant enzymes were highly active on artificial (p-nitrophenyl ß-D-xylopyranoside) and natural (xylobiose, xylotriose, and xylotetraose) substrates, and showed transxylosylation activity and tolerance to xylose inhibition. The enzymes belong to glycoside hydrolase family 120 with Asp as nucleophile and Glu as proton donor, as shown by homology modelling and confirmed by site-directed mutagenesis. In silico analysis revealed that these enzymes were part of a gene cluster in L. reuteri but not in Blautia strains, and quantitative proteomics identified other enzymes and transporters involved in B. producta XOS utilisation. Based on these findings, we proposed a model for an XOS metabolism pathway in L. reuteri and B. producta strains. Together with phylogenetic analyses, the data also revealed the extended xylanolytic potential of the gut microbiota.


Asunto(s)
Xilanos , Xilosidasas , Bacterias/genética , Bacterias/metabolismo , Glucuronatos , Humanos , Oligosacáridos , Filogenia , Especificidad por Sustrato , Xilanos/metabolismo , Xilosidasas/metabolismo
6.
J Biol Chem ; 295(52): 18625-18637, 2020 12 25.
Artículo en Inglés | MEDLINE | ID: mdl-33097594

RESUMEN

Pectins are a major dietary nutrient source for the human gut microbiota. The prominent gut microbe Bacteroides thetaiotaomicron was recently shown to encode the founding member (BT1017) of a new family of pectin methylesterases essential for the metabolism of the complex pectin rhamnogalacturonan-II (RG-II). However, biochemical and structural knowledge of this family is lacking. Here, we showed that BT1017 is critical for the metabolism of an RG-II-derived oligosaccharide ΔBT1017oligoB generated by a BT1017 deletion mutant (ΔBT1017) during growth on carbohydrate extract from apple juice. Structural analyses of ΔBT1017oligoB using a combination of enzymatic, mass spectrometric, and NMR approaches revealed that it is a bimethylated nonaoligosaccharide (GlcA-ß1,4-(2-O-Me-Xyl-α1,3)-Fuc-α1,4-(GalA-ß1,3)-Rha-α1,3-Api-ß1,2-(Araf-α1,3)-(GalA-α1,4)-GalA) containing components of the RG-II backbone and its side chains. We showed that the catalytic module of BT1017 adopts an α/ß-hydrolase fold, consisting of a central twisted 10-stranded ß-sheet sandwiched by several α-helices. This constitutes a new fold for pectin methylesterases, which are predominantly right-handed ß-helical proteins. Bioinformatic analyses revealed that the family is dominated by sequences from prominent genera of the human gut microbiota, including Bacteroides and Prevotella Our re-sults not only highlight the critical role played by this family of enzymes in pectin metabolism but also provide new insights into the molecular basis of the adaptation of B. thetaiotaomicron to the human gut.


Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Bacteroides thetaiotaomicron/enzimología , Hidrolasas de Éster Carboxílico/química , Hidrolasas de Éster Carboxílico/metabolismo , Microbioma Gastrointestinal , Oligosacáridos/metabolismo , Bacteroides thetaiotaomicron/crecimiento & desarrollo , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Filogenia , Conformación Proteica
7.
Chemistry ; 27(4): 1374-1382, 2021 Jan 18.
Artículo en Inglés | MEDLINE | ID: mdl-32990374

RESUMEN

Understanding the fine details of the self-assembly of building blocks into complex hierarchical structures represents a major challenge en route to the design and preparation of soft-matter materials with specific properties. Enzymatically synthesised cellodextrins are known to have limited water solubility beyond DP9, a point at which they self-assemble into particles resembling the antiparallel cellulose II crystalline packing. We have prepared and characterised a series of site-selectively fluorinated cellodextrins with different degrees of fluorination and substitution patterns by chemoenzymatic synthesis. Bearing in mind the potential disruption of the hydrogen-bond network of cellulose II, we have prepared and characterised a multiply 6-fluorinated cellodextrin. In addition, a series of single site-selectively fluorinated cellodextrins was synthesised to assess the structural impact upon the addition of one fluorine atom per chain. The structural characterisation of these materials at different length scales, combining advanced NMR spectroscopy and microscopy methods, showed that a 6-fluorinated donor substrate yielded multiply 6-fluorinated cellodextrin chains that assembled into particles presenting morphological and crystallinity features, and intermolecular interactions, that are unprecedented for cellulose-like materials.

8.
J Biol Chem ; 294(19): 7711-7721, 2019 05 10.
Artículo en Inglés | MEDLINE | ID: mdl-30877196

RESUMEN

The metabolism of carbohydrate polymers drives microbial diversity in the human gut microbiome. The selection pressures in this environment have spurred the evolution of a complex reservoir of microbial genes encoding carbohydrate-active enzymes (CAZymes). Previously, we have shown that the human gut bacterium Bacteroides thetaiotaomicron (Bt) can depolymerize the most structurally complex glycan, the plant pectin rhamnogalacturonan II (RGII), commonly found in the human diet. Previous investigation of the RGII-degrading apparatus in Bt identified BT0997 as a new CAZyme family, classified as glycoside hydrolase 138 (GH138). The mechanism of substrate recognition by GH138, however, remains unclear. Here, using synthetic substrates and biochemical assays, we show that BT0997 targets the d-galacturonic acid-α-1,2-l-rhamnose linkage in chain A of RGII and that it absolutely requires the presence of a second d-galacturonic acid side chain (linked ß-1,3 to l-rhamnose) for activity. NMR analysis revealed that BT0997 operates through a double displacement retaining mechanism. We also report the crystal structure of a BT0997 homolog, BPA0997 from Bacteroides paurosaccharolyticus, in complex with ligands at 1.6 Å resolution. The structure disclosed that the enzyme comprises four domains, including a catalytic TIM (α/ß)8 barrel. Characterization of several BT0997 variants identified Glu-294 and Glu-361 as the catalytic acid/base and nucleophile, respectively, and we observed a chloride ion close to the active site. The three-dimensional structure and bioinformatic analysis revealed that two arginines, Arg-332 and Arg-521, are key specificity determinants of BT0997 in targeting d-galacturonic acid residues. In summary, our study reports the first structural and mechanistic analyses of GH138 enzymes.


Asunto(s)
Proteínas Bacterianas/química , Bacteroides thetaiotaomicron/enzimología , Glicósido Hidrolasas/química , Ácidos Hexurónicos/química , Proteínas Bacterianas/genética , Bacteroides thetaiotaomicron/genética , Dominio Catalítico , Cristalografía por Rayos X , Glicósido Hidrolasas/genética , Relación Estructura-Actividad , Especificidad por Sustrato
9.
J Biol Chem ; 294(23): 9172-9185, 2019 06 07.
Artículo en Inglés | MEDLINE | ID: mdl-31010825

RESUMEN

The 6-deoxy sugar l-rhamnose (l-Rha) is found widely in plant and microbial polysaccharides and natural products. The importance of this and related compounds in host-pathogen interactions often means that l-Rha plays an essential role in many organisms. l-Rha is most commonly biosynthesized as the activated sugar nucleotide uridine 5'-diphospho-ß-l-rhamnose (UDP-ß-l-Rha) or thymidine 5'-diphospho-ß-l-rhamnose (TDP-ß-l-Rha). Enzymes involved in the biosynthesis of these sugar nucleotides have been studied in some detail in bacteria and plants, but the activated form of l-Rha and the corresponding biosynthetic enzymes have yet to be explored in algae. Here, using sugar-nucleotide profiling in two representative algae, Euglena gracilis and the toxin-producing microalga Prymnesium parvum, we show that levels of UDP- and TDP-activated l-Rha differ significantly between these two algal species. Using bioinformatics and biochemical methods, we identified and characterized a fusion of the RmlC and RmlD proteins, two bacteria-like enzymes involved in TDP-ß-l-Rha biosynthesis, from P. parvum Using this new sequence and also others, we explored l-Rha biosynthesis among algae, finding that although most algae contain sequences orthologous to plant-like l-Rha biosynthesis machineries, instances of the RmlC-RmlD fusion protein identified here exist across the Haptophyta and Gymnodiniaceae families of microalgae. On the basis of these findings, we propose potential routes for the evolution of nucleoside diphosphate ß-l-Rha (NDP-ß-l-Rha) pathways among algae.


Asunto(s)
Proteínas Algáceas/metabolismo , Carbohidrato Epimerasas/metabolismo , Haptophyta/metabolismo , Ramnosa/biosíntesis , Proteínas Algáceas/genética , Carbohidrato Epimerasas/clasificación , Carbohidrato Epimerasas/genética , Filogenia , Plastidios/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Ramnosa/química , Simbiosis
10.
Chembiochem ; 21(7): 1043-1049, 2020 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-31657512

RESUMEN

The enzymatic synthesis of oligosaccharides depends on the availability of suitable enzymes, which remains a limitation. Without recourse to enzyme engineering or evolution approaches, herein we demonstrate the ability of wild-type cellodextrin phosphorylase (CDP: ß-1,4-glucan linkage-dependent) and laminaridextrin phosphorylase (Pro_7066: ß-1,3-glucan linkage-dependent) to tolerate a number of sugar-1- phosphate substrates, albeit with reduced kinetic efficiency. In spite of catalytic efficiencies of <1 % of the natural reactions, we demonstrate the utility of given phosphorylase-sugar phosphate pairs to access new-to-nature fragments of human milk oligosaccharides, or analogues thereof, in multi-milligram quantities.


Asunto(s)
Leche Humana/metabolismo , Oligosacáridos/metabolismo , Fosforilasas/metabolismo , Biocatálisis , Dominio Catalítico , Glucosiltransferasas/metabolismo , Humanos , Cinética , Simulación de Dinámica Molecular , Oligosacáridos/química , Especificidad por Sustrato
11.
Chembiochem ; 20(2): 181-192, 2019 01 18.
Artículo en Inglés | MEDLINE | ID: mdl-29856496

RESUMEN

Glycoside phosphorylases (GPs) carry out a reversible phosphorolysis of carbohydrates into oligosaccharide acceptors and the corresponding sugar 1-phosphates. The reversibility of the reaction enables the use of GPs as biocatalysts for carbohydrate synthesis. Glycosyl hydrolase family 94 (GH94), which only comprises GPs, is one of the most studied GP families that have been used as biocatalysts for carbohydrate synthesis, in academic research and in industrial production. Understanding the mechanism of GH94 enzymes is a crucial step towards enzyme engineering to improve and expand the applications of these enzymes in synthesis. In this work with a GH94 laminaribiose phosphorylase from Paenibacillus sp. YM-1 (PsLBP), we have demonstrated an enzymatic synthesis of disaccharide 1 (ß-d-mannopyranosyl-(1→3)-d-glucopyranose) by using a natural acceptor glucose and noncognate donor substrate α-mannose 1-phosphate (Man1P). To investigate how the enzyme recognises different sugar 1-phosphates, the X-ray crystal structures of PsLBP in complex with Glc1P and Man1P have been solved, providing the first molecular detail of the recognition of a noncognate donor substrate by GPs, which revealed the importance of hydrogen bonding between the active site residues and hydroxy groups at C2, C4, and C6 of sugar 1-phosphates. Furthermore, we used saturation transfer difference NMR spectroscopy to support crystallographic studies on the sugar 1-phosphates, as well as to provide further insights into the PsLBP recognition of the acceptors and disaccharide products.


Asunto(s)
Glucosa/química , Glucosiltransferasas/química , Manosafosfatos/química , Paenibacillus/enzimología , Cristalografía por Rayos X , Glucosa/metabolismo , Glucosiltransferasas/metabolismo , Manosafosfatos/metabolismo , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Especificidad por Sustrato
12.
J Biol Chem ; 290(50): 29834-53, 2015 Dec 11.
Artículo en Inglés | MEDLINE | ID: mdl-26504082

RESUMEN

The degradation of transitory starch in the chloroplast to provide fuel for the plant during the night requires a suite of enzymes that generate a series of short chain linear glucans. However, glucans of less than four glucose units are no longer substrates for these enzymes, whereas export from the plastid is only possible in the form of either maltose or glucose. In order to make use of maltotriose, which would otherwise accumulate, disproportionating enzyme 1 (DPE1; a 4-α-glucanotransferase) converts two molecules of maltotriose to a molecule of maltopentaose, which can now be acted on by the degradative enzymes, and one molecule of glucose that can be exported. We have determined the structure of the Arabidopsis plastidial DPE1 (AtDPE1), and, through ligand soaking experiments, we have trapped the enzyme in a variety of conformational states. AtDPE1 forms a homodimer with a deep, long, and open-ended active site canyon contained within each subunit. The canyon is divided into donor and acceptor sites with the catalytic residues at their junction; a number of loops around the active site adopt different conformations dependent on the occupancy of these sites. The "gate" is the most dynamic loop and appears to play a role in substrate capture, in particular in the binding of the acceptor molecule. Subtle changes in the configuration of the active site residues may prevent undesirable reactions or abortive hydrolysis of the covalently bound enzyme-substrate intermediate. Together, these observations allow us to delineate the complete AtDPE1 disproportionation cycle in structural terms.


Asunto(s)
Arabidopsis/enzimología , Enzimas/metabolismo , Plastidios/enzimología , Polisacáridos/metabolismo , Secuencia de Aminoácidos , Cristalografía por Rayos X , Enzimas/química , Datos de Secuencia Molecular , Conformación Proteica , Homología de Secuencia de Aminoácido
13.
Tetrahedron ; 71(39): 7344-7353, 2015 Sep 30.
Artículo en Inglés | MEDLINE | ID: mdl-26435551

RESUMEN

Reaction of 2-(2-(2-azidoethoxy)ethoxy)ethyl 6-O-(prop-2-ynyl)-ß-d-galactopyranoside (7) under CuAAC conditions gives rise to mixed cyclic and linear triazole-linked oligomers, with individual compounds up to d.p. 5 isolable, along with mixed larger oligomers. The linear compounds resolve en bloc from the cyclic materials by RP HPLC, but are separable by gel permeation chromatography. The triazole-linked oligomers-pseudo-galactooligomers-were demonstrated to be acceptor substrates for the multi-copy cell surface trans-sialidase of the human parasite Trypanosoma cruzi. In addition, these multivalent TcTS ligands were able to block macrophage invasion by T. cruzi.

14.
Carbohydr Res ; 528: 108807, 2023 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-37094534

RESUMEN

ß-(1,2)-Mannan antigens incorporated into vaccines candidates for immunization studies, showed that antibodies raised against ß-(1,2)-mannotriose antigens can protect against disseminated candidiasis. Until recently, ß-(1,2)- mannans could only be obtained by isolation from microbial cultures, or by lengthy synthetic strategies involving protecting group manipulation. The discovery of two ß-(1,2)-mannoside phosphorylases, Teth514_1788 and Teth514_1789, allowed efficient access to these compounds. In this work, Teth514_1788 was utilised to generate ß-(1,2)-mannan antigens, tri- and tetra-saccharides, decorated with a conjugation tether at the reducing end, suitable to be incorporated on a carrier en-route to novel vaccine candidates, illustrated here by conjugation of the trisaccharide to BSA.


Asunto(s)
Candidiasis , Glucógeno Fosforilasa de Forma Muscular , Humanos , Mananos , Candidiasis/prevención & control , Oligosacáridos , Fosforilasas , Vacunas Conjugadas
15.
Carbohydr Polym ; 277: 118606, 2022 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-34893207

RESUMEN

Levan, a ß-2,6 fructofuranose polymer produced by microbial species, has been reported for its immunomodulatory properties via interaction with toll-like receptor 4 (TLR4) which recognises lipopolysaccharide (LPS). However, the molecular mechanisms underlying these interactions remain elusive. Here, we investigated the immunomodulatory properties of levan using thoroughly-purified and characterised samples from Erwinia herbicola and other sources. E. herbicola levan was purified by gel-permeation chromatography and LPS was removed from the levan following a novel alkali treatment developed in this study. E. herbicola levan was then characterised by gas chromatography-mass spectrometry and NMR. We found that levan containing LPS, but not LPS-depleted levan, induced TLR4-mediated cytokine production by bone marrow-derived dendritic cells and/or activated TLR4 reporter cells. These data indicated that the immunomodulatory properties of the levan toward TLR4-expressing immune cells were mediated by the LPS. This work also demonstrates the importance of LPS removal when assessing the immunomodulatory activity of polysaccharides.


Asunto(s)
Fructanos/farmacología , Factores Inmunológicos/farmacología , Lipopolisacáridos/farmacología , Receptor Toll-Like 4/inmunología , Animales , Línea Celular , Citocinas/biosíntesis , Erwinia/química , Fructanos/química , Humanos , Factores Inmunológicos/química , Lipopolisacáridos/química , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor Toll-Like 4/deficiencia
16.
Org Biomol Chem ; 9(19): 6670-84, 2011 Oct 07.
Artículo en Inglés | MEDLINE | ID: mdl-21847487

RESUMEN

Fragments of pectic polysaccharides rhamnogalacturonan-II (RG-II) and apiogalacturonan were synthesised using p-tolylthio apiofuranoside derivatives as key building blocks. Apiofuranose thioglycosides can be conveniently prepared by cyclization of the corresponding dithioacetals possessing a 2,3-O-isopropylidene group, which is required for preservation of the correct (3R) configuration of the apiofuranose ring. The remarkable stability of this protecting group in apiofuranose derivatives requires its replacement with a more reactive protecting group, such as a benzylidene acetal which was used in the synthesis of trisaccharide ß-Rhap-(1→3')-ß-Apif-(1→2)-α-GalAp-OMe. The X-ray crystal structure of the protected precursor of this trisaccharide has been elucidated.


Asunto(s)
Araceae/química , Pectinas/síntesis química , Pentosas/química , Zosteraceae/química , Araceae/citología , Conformación de Carbohidratos , Cristalografía por Rayos X , Modelos Moleculares , Pectinas/química , Zosteraceae/citología
17.
Int J Biol Macromol ; 187: 690-698, 2021 Sep 30.
Artículo en Inglés | MEDLINE | ID: mdl-34343579

RESUMEN

Fermentation of Lactic Acid Bacteria (LAB) is considered to be a sustainable approach for polysaccharide production. Herein, exopolysaccharide (EPS)-producing LAB strain KM01 was isolated from Thai fermented dessert, Khao Mak, which was then identified as Leuconostoc holzapfelii. High-performance anion-exchange chromatography, nuclear magnetic resonance spectroscopy and Fourier-transform infrared spectroscopy suggested that the KM01 EPS comprises α-1,6-linked glucosides. The molecular weight of KM01 EPS was around 500 kDa, but it can form large aggregates formation (MW > 2000 kDa) in an aqueous solution, judged by transmission electron microscopy and dynamic light scattering to be around 150 nm in size. Furthermore, this KM01 EPS form highly viscous hydrogels at concentrations above 5% (w/v). The formation of hydrogels and nanoparticle of KM01 EPS was found to be reversible. Finally, the suitability of KM01 EPS for biomedical applications was demonstrated by its lack of cytotoxicity and its ability to form complexes with quercetin. Unlike the common α-1,6-linked dextran, KM01 EPS can enhance the solubility of quercetin significantly.


Asunto(s)
Excipientes/química , Glucanos/química , Leuconostoc/metabolismo , Nanopartículas , Polisacáridos Bacterianos/química , Quercetina/química , Sacarosa/metabolismo , Composición de Medicamentos , Excipientes/aislamiento & purificación , Excipientes/toxicidad , Fermentación , Glucanos/aislamiento & purificación , Glucanos/toxicidad , Hidrogeles , Peso Molecular , Polisacáridos Bacterianos/aislamiento & purificación , Polisacáridos Bacterianos/toxicidad , Solubilidad , Viscosidad
18.
Elife ; 102021 12 31.
Artículo en Inglés | MEDLINE | ID: mdl-34792466

RESUMEN

Agricultural soil harbors a diverse microbiome that can form beneficial relationships with plants, including the inhibition of plant pathogens. Pseudomonas spp. are one of the most abundant bacterial genera in the soil and rhizosphere and play important roles in promoting plant health. However, the genetic determinants of this beneficial activity are only partially understood. Here, we genetically and phenotypically characterize the Pseudomonas fluorescens population in a commercial potato field, where we identify strong correlations between specialized metabolite biosynthesis and antagonism of the potato pathogens Streptomyces scabies and Phytophthora infestans. Genetic and chemical analyses identified hydrogen cyanide and cyclic lipopeptides as key specialized metabolites associated with S. scabies inhibition, which was supported by in planta biocontrol experiments. We show that a single potato field contains a hugely diverse and dynamic population of Pseudomonas bacteria, whose capacity to produce specialized metabolites is shaped both by plant colonization and defined environmental inputs.


Potato scab and blight are two major diseases which can cause heavy crop losses. They are caused, respectively, by the bacterium Streptomyces scabies and an oomycete (a fungus-like organism) known as Phytophthora infestans. Fighting these disease-causing microorganisms can involve crop management techniques ­ for example, ensuring that a field is well irrigated helps to keep S. scabies at bay. Harnessing biological control agents can also offer ways to control disease while respecting the environment. Biocontrol bacteria, such as Pseudomonas, can produce compounds that keep S. scabies and P. infestans in check. However, the identity of these molecules and how irrigation can influence Pseudomonas population remains unknown. To examine these questions, Pacheco-Moreno et al. sampled and isolated hundreds of Pseudomonas strains from a commercial potato field, closely examining the genomes of 69 of these. Comparing the genetic information of strains based on whether they could control the growth of S. scabies revealed that compounds known as cyclic lipopeptides are key to controlling the growth of S. scabies and P. infestans. Whether the field was irrigated also had a large impact on the strains forming the Pseudomonas population. Working out how Pseudomonas bacteria block disease could speed up the search for biological control agents. The approach developed by Pacheco-Moreno et al. could help to predict which strains might be most effective based on their genetic features. Similar experiments could also work for other combinations of plants and diseases.


Asunto(s)
Phytophthora infestans/fisiología , Enfermedades de las Plantas/microbiología , Pseudomonas fluorescens/genética , Solanum tuberosum/microbiología , Streptomyces/fisiología , Cianuro de Hidrógeno/metabolismo , Lipopéptidos/metabolismo , Péptidos Cíclicos/metabolismo , Pseudomonas fluorescens/metabolismo
19.
Bioorg Med Chem ; 18(7): 2412-27, 2010 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-20335038

RESUMEN

Trypanosoma cruzi trans-sialidase (TcTS) plays a key role in the recognition and invasion of host cells and in enabling the parasite to escape the human immune response. To explore this potential drug target, we have synthesized a small library of substrate analogues based on 1,4-disubstituted 1,2,3-triazole derivatives of galactose modified at either the C-1 or C-6 positions. This was achieved by coupling the appropriate azido-sugars with a panel of 23 structurally diverse terminal alkynes by using the copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) reaction, giving a library of 46 derivatives in good to excellent yield and with complete regioselectivity. The sugar triazoles showed weak inhibition towards TcTS-catalyzed hydrolysis of 2'-(4-methylumbelliferyl)-alpha-d-N-acetylneuraminic acid in vitro (<40% inhibition at 1mM concentration); many of the compounds assessed proved to be acceptor substrates for the enzyme. Despite this modest inhibitory activity, in vitro trypanocidal activity assays against the trypomastigote form of T. cruzi Y strain revealed several compounds active in the low 100s of muM range. Further assessment of these compounds against cultured mouse spleen cells suggests a specific mode of anti-parasite action rather than a generic cytotoxic effect.


Asunto(s)
Galactosa/análogos & derivados , Galactosa/síntesis química , Neuraminidasa/antagonistas & inhibidores , Triazoles/síntesis química , Triazoles/farmacología , Tripanocidas/síntesis química , Tripanocidas/farmacología , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/enzimología , Animales , Azidas/síntesis química , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cromatografía en Capa Delgada , Fluorometría , Galactosa/farmacología , Indicadores y Reactivos , Espectroscopía de Resonancia Magnética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Espectrofotometría Ultravioleta , Bazo/citología , Bazo/efectos de los fármacos , Relación Estructura-Actividad
20.
Int J Biol Macromol ; 152: 473-482, 2020 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-32097735

RESUMEN

Glucansucrases catalyse the formation of glucans from sucrose. The glucansucrase-encoding gene from Leuconostoc citreum ABK-1, dex-N, was successfully cloned and expressed in E. coli BL21 Star (DE3). DEX-N produces 2 types of glucans: soluble (S-dextran) and insoluble (I-glucan) glucans. The S-dextran was determined to be ca. 10 kDa in size and contained >90% α-1,6 linkages; along with its water solubility, this is similar to commercial dextran. On the other hand, I-glucan was water-insoluble, harbouring a block-wise pattern of α-1,3 and α-1,6 linkages in its structure. Notably, the FTIR and powder X-ray diffraction pattern of I-glucan exhibited a combination of features found in α-1,6-linked dextran and α-1,3-linked mutan. Although both I-glucan and mutan are insoluble glucans, their physical characteristics are notably dissimilar.


Asunto(s)
Proteínas Bacterianas/química , Dextranos/química , Glucanos/química , Glicosiltransferasas/química , Leuconostoc/enzimología , Clonación Molecular , Escherichia coli , Concentración de Iones de Hidrógeno , Iones , Espectroscopía de Resonancia Magnética , Metales , Metilación , Peso Molecular , Solubilidad , Espectroscopía Infrarroja por Transformada de Fourier , Streptococcus mutans , Temperatura , Viscosidad , Difracción de Rayos X
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