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1.
Biochemistry ; 61(15): 1572-1584, 2022 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-35861590

RESUMO

Glycosyltransferase (GT) enzymes promote the formation of glycosidic bonds between a sugar molecule and a diversity of substrates. Heptosyltransferase II (HepII) is a GT involved in the lipopolysaccharide (LPS) biosynthetic pathway that transfers the seven-carbon sugar (l-glycero-d-manno-heptose, Hep) onto a lipid-anchored glycopolymer (heptosylated Kdo2-Lipid A, Hep-Kdo2-Lipid A, or HLA). LPS plays a key role in Gram-negative bacterial sepsis, biofilm formation, and host colonization, and as such, LPS biosynthetic enzymes are targets for novel antimicrobial therapeutics. Three heptosyltransferases are involved in the inner-core LPS biosynthesis, with Escherichia coli HepII being the last to be quantitatively characterized in vivo. HepII shares modest sequence similarity with heptosyltransferase I (HepI) while maintaining a high degree of structural homology. Here, we report the first kinetic and biophysical characterization of HepII and demonstrate the properties of HepII that are shared with HepI, including sugar donor promiscuity and sugar acceptor-induced secondary structural changes, which results in significant thermal stabilization. HepII also has an increased catalytic efficiency and a significantly tighter binding affinity for both of its substrates compared to HepI. A structural model of the HepII ternary complex, refined by molecular dynamics simulations, was developed to probe the potentially important substrate-protein contacts. Ligand binding-induced changes in Trp fluorescence in HepII enabled the determination of substrate dissociation constants. Combined, these efforts meaningfully enhance our understanding of the heptosyltransferase family of enzymes and will aid in future efforts to design novel, potent, and specific inhibitors for this family of enzymes.


Assuntos
Escherichia coli , Glicosiltransferases , Lipídeo A , Catálise , Escherichia coli/enzimologia , Glicosiltransferases/metabolismo , Heptoses/química , Lipídeo A/metabolismo , Lipopolissacarídeos , Simulação de Dinâmica Molecular
2.
Int J Mol Sci ; 23(11)2022 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-35682658

RESUMO

Glioblastoma (GBM) is the most common form of malignant brain cancer and is considered the deadliest human cancer. Because of poor outcomes in this disease, there is an urgent need for progress in understanding the molecular mechanisms of GBM therapeutic resistance, as well as novel and innovative therapies for cancer prevention and treatment. The pentose phosphate pathway (PPP) is a metabolic pathway complementary to glycolysis, and several PPP enzymes have already been demonstrated as potential targets in cancer therapy. In this work, we aimed to evaluate the role of sedoheptulose kinase (SHPK), a key regulator of carbon flux that catalyzes the phosphorylation of sedoheptulose in the nonoxidative arm of the PPP. SHPK expression was investigated in patients with GBM using microarray data. SHPK was also overexpressed in GBM cells, and functional studies were conducted. SHPK expression in GBM shows a significant correlation with histology, prognosis, and survival. In particular, its increased expression is associated with a worse prognosis. Furthermore, its overexpression in GBM cells confirms an increase in cell proliferation. This work highlights for the first time the importance of SHPK in GBM for tumor progression and proposes this enzyme and the nonoxidative PPP as possible therapeutic targets.


Assuntos
Glioblastoma , Via de Pentose Fosfato , Proliferação de Células , Glioblastoma/genética , Glioblastoma/metabolismo , Heptoses , Humanos
3.
Biochemistry ; 61(13): 1313-1322, 2022 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-35715226

RESUMO

Campylobacter jejuni is a human pathogen and a leading cause of food poisoning in the United States and Europe. Surrounding the outside of the bacterium is a carbohydrate coat known as the capsular polysaccharide. Various strains of C. jejuni have different sequences of unusual sugars and an assortment of decorations. Many of the serotypes have heptoses with differing stereochemical arrangements at C2 through C6. One of the many common modifications is a 6-deoxy-heptose that is formed by dehydration of GDP-d-glycero-α-d-manno-heptose to GDP-6-deoxy-4-keto-d-lyxo-heptose via the action of the enzyme GDP-d-glycero-α-d-manno-heptose 4,6-dehydratase. Herein, we report the biochemical and structural characterization of this enzyme from C. jejuni 81-176 (serotype HS:23/36). The enzyme was purified to homogeneity, and its three-dimensional structure was determined to a resolution of 2.1 Å. Kinetic analyses suggest that the reaction mechanism proceeds through the formation of a 4-keto intermediate followed by the loss of water from C5/C6. Based on the three-dimensional structure, it is proposed that oxidation of C4 is assisted by proton transfer from the hydroxyl group to the phenolate of Tyr-159 and hydride transfer to the tightly bound NAD+ in the active site. Elimination of water at C5/C6 is most likely assisted by abstraction of the proton at C5 by Glu-136 and subsequent proton transfer to the hydroxyl at C6 via Ser-134 and Tyr-159. A bioinformatic analysis identified 19 additional 4,6-dehydratases from serotyped strains of C. jejuni that are 89-98% identical in the amino acid sequence, indicating that each of these strains should contain a 6-deoxy-heptose within their capsular polysaccharides.


Assuntos
Campylobacter jejuni , Proteínas de Bactérias/química , Heptoses/química , Humanos , Hidroliases/metabolismo , Prótons , Água/metabolismo
4.
Infect Immun ; 90(7): e0022422, 2022 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-35762751

RESUMO

Klebsiella pneumoniae is a leading cause of Gram-negative bacteremia, which is a major source of morbidity and mortality worldwide. Gram-negative bacteremia requires three major steps: primary site infection, dissemination to the blood, and bloodstream survival. Because K. pneumoniae is a leading cause of health care-associated pneumonia, the lung is a common primary infection site leading to secondary bacteremia. K. pneumoniae factors essential for lung fitness have been characterized, but those required for subsequent bloodstream infection are unclear. To identify K. pneumoniae genes associated with dissemination and bloodstream survival, we combined previously and newly analyzed insertion site sequencing (InSeq) data from a murine model of bacteremic pneumonia. This analysis revealed the gene gmhB as important for either dissemination from the lung or bloodstream survival. In Escherichia coli, GmhB is a partially redundant enzyme in the synthesis of ADP-heptose for the lipopolysaccharide (LPS) core. To characterize its function in K. pneumoniae, an isogenic knockout strain (ΔgmhB) and complemented mutant were generated. During pneumonia, GmhB did not contribute to lung fitness and did not alter normal immune responses. However, GmhB enhanced bloodstream survival in a manner independent of serum susceptibility, specifically conveying resistance to spleen-mediated killing. In a tail-vein injection of murine bacteremia, GmhB was also required by K. pneumoniae, E. coli, and Citrobacter freundii for optimal fitness in the spleen and liver. Together, this study identifies GmhB as a conserved Gram-negative bacteremia fitness factor that acts through LPS-mediated mechanisms to enhance fitness in blood-filtering organs.


Assuntos
Bacteriemia , Infecções por Klebsiella , Difosfato de Adenosina , Animais , Bacteriemia/genética , Escherichia coli/genética , Heptoses , Klebsiella pneumoniae/genética , Lipopolissacarídeos , Camundongos
5.
Plant Physiol Biochem ; 171: 49-65, 2022 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-34971955

RESUMO

The mechanism of the combined action of potassium (K) and melatonin (Mel) in modulating tolerance to cadmium (Cd) stress in plants is not well understood. The present study reveals the synergistic role of K and Mel in enhancing physiological and biochemical mechanisms of Cd stress tolerance in tomato seedlings. The present findings reveal that seedlings subjected to Cd toxicity exhibited disturbed nutrients balance [nitrogen (N) and potassium (K)], chlorophyll (Chl) biosynthesis [reduced δ-aminolevulinic acid (δ-ALA) content and δ-aminolevulinic acid dehydratase (δ-ALAD) activity], pathway of carbon fixation [reduced fructose-1,6-bisphosphatase (FBPase) and sedoheptulose-1,7- bisphosphatase (SBPase) activity] and photosynthesis process in tomato seedlings. However, exogenous application of K and Mel alone as well as together improved physiological and biochemical mechanisms in tomato seedlings, but their combined application proved best by efficiently improving nutrient uptake, photosynthetic pigments biosynthesis (increased Chl a and b, and Total Chl), carbon flow in Calvin cycle, activity of Rubisco, carbonic anhydrase activity, and accumulation of total soluble carbohydrates content in seedlings under Cd toxicity. Furthermore, the combined treatment of K and Mel suppressed overproduction of reactive oxygen species (hydrogen peroxide and superoxide), Chl degradation [reduced chlorophyllase (Chlase) activity] and methylglyoxal content in Cd-stressed tomato seedlings by upregulating glyoxalase (increased glyoxalase I and glyoxalase II activity) and antioxidant systems (increased ascorbate-glutathione metabolism). Thus, the present study provides stronger evidence that the co-application of K and Mel exhibited synergistic roles in mitigating the toxic effect of Cd stress by increasing glyoxalase and antioxidant systems and also by improving photosynthetic efficiency in tomato seedlings.


Assuntos
Lycopersicon esculentum , Melatonina , Antioxidantes/metabolismo , Cádmio/toxicidade , Carbono , Frutose , Frutose-Bifosfatase , Heptoses , Lycopersicon esculentum/metabolismo , Fotossíntese , Potássio , Plântula/metabolismo
6.
Biochemistry ; 60(37): 2836-2843, 2021 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-34505775

RESUMO

Campylobacter jejuni is a Gram-negative, pathogenic bacterium that causes campylobacteriosis, a form of gastroenteritis. C. jejuni is the most frequent cause of food-borne illness in the world, surpassing Salmonella and E. coli. Coating the surface of C. jejuni is a layer of sugar molecules known as the capsular polysaccharide that, in C. jejuni NCTC 11168, is composed of a repeating unit of d-glycero-l-gluco-heptose, d-glucuronic acid, d-N-acetyl-galactosamine, and d-ribose. The d-glucuronic acid moiety is further amidated with either serinol or ethanolamine. It is unknown how these modifications are synthesized and attached to the polysaccharide. Here, we report the catalytic activities of two previously uncharacterized, pyridoxal phosphate (PLP)-dependent enzymes, Cj1436 and Cj1437, from C. jejuni NCTC 11168. Using a combination of mass spectrometry and nuclear magnetic resonance, we determined that Cj1436 catalyzes the decarboxylation of l-serine phosphate to ethanolamine phosphate. Cj1437 was shown to catalyze the transamination of dihydroxyacetone phosphate to (S)-serinol phosphate in the presence of l-glutamate. The probable routes to the ultimate formation of the glucuronamide substructures in the capsular polysaccharides of C. jejuni are discussed.


Assuntos
Cápsulas Bacterianas/enzimologia , Cápsulas Bacterianas/metabolismo , Campylobacter jejuni/enzimologia , Cápsulas Bacterianas/genética , Proteínas de Bactérias/química , Infecções por Campylobacter/microbiologia , Campylobacter jejuni/metabolismo , Metabolismo dos Carboidratos , Heptoses/biossíntese , Polissacarídeos/biossíntese , Polissacarídeos Bacterianos/biossíntese , Polissacarídeos Bacterianos/genética , Polissacarídeos Bacterianos/metabolismo , Fosfato de Piridoxal/metabolismo
7.
FEBS Lett ; 595(16): 2160-2168, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34216493

RESUMO

The persistence of Helicobacter pylori in the human gastric mucosa implies that the immune response fails to clear the infection. We found that H. pylori compromises the antigen presentation ability of macrophages, because of the decline of the presenting molecules HLA-II. Here, we reveal that the main bacterial factor responsible for this effect is ADP-heptose, an intermediate metabolite in the biosynthetic pathway of lipopolysaccharide (LPS) that elicits a pro-inflammatory response in gastric epithelial cells. In macrophages, it upregulates the expression of miR146b which, in turn, would downmodulate CIITA, the master regulator for HLA-II genes. Hence, H. pylori, utilizing ADP-heptose, exploits a specific arm of macrophage response to establish its survival niche in the face of the immune defense elicited in the gastric mucosa.


Assuntos
Apresentação de Antígeno/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Helicobacter pylori/fisiologia , Heptoses/farmacologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Macrófagos/efeitos dos fármacos , Helicobacter pylori/metabolismo , Heptoses/química , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Proteínas Nucleares/metabolismo , Transativadores/metabolismo
8.
Front Immunol ; 12: 632154, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34093525

RESUMO

The human gastric pathogen Helicobacter pylori activates human epithelial cells by a particular combination of mechanisms, including NOD1 and ALPK1-TIFA activation. These mechanisms are characterized by a strong participation of the bacterial cag pathogenicity island, which forms a type IV secretion system (CagT4SS) that enables the bacteria to transport proteins and diverse bacterial metabolites, including DNA, glycans, and cell wall components, into human host cells. Building on previous findings, we sought to determine the contribution of lipopolysaccharide inner core heptose metabolites (ADP-heptose) in the activation of human phagocytic cells by H. pylori. Using human monocyte/macrophage-like Thp-1 cells and human primary monocytes and macrophages, we were able to determine that a substantial part of early phagocytic cell activation, including NF-κB activation and IL-8 production, by live H. pylori is triggered by bacterial heptose metabolites. This effect was very pronounced in Thp-1 cells exposed to bacterial purified lysates or pure ADP-heptose, in the absence of other bacterial MAMPs, and was significantly reduced upon TIFA knock-down. Pure ADP-heptose on its own was able to strongly activate Thp-1 cells and human primary monocytes/macrophages. Comprehensive transcriptome analysis of Thp-1 cells co-incubated with live H. pylori or pure ADP-heptose confirmed a signature of ADP-heptose-dependent transcript activation in monocyte/macrophages. Bacterial enzyme-treated lysates (ETL) and pure ADP-heptose-dependent activation differentiated monocytes into macrophages of predominantly M1 type. In Thp-1 cells, the active CagT4SS was less required for the heptose-induced proinflammatory response than in epithelial cells, while active heptose biosynthesis or pure ADP-heptose was required and sufficient for their early innate response and NF-κB activation. The present data suggest that early activation and maturation of incoming and resident phagocytic cells (monocytes, macrophages) in the H. pylori-colonized stomach strongly depend on bacterial LPS inner core heptose metabolites, also with a significant contribution of an active CagT4SS.


Assuntos
Ilhas Genômicas/fisiologia , Helicobacter pylori/metabolismo , Heptoses/metabolismo , Macrófagos/imunologia , Monócitos/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Vias Biossintéticas , Helicobacter pylori/patogenicidade , Humanos , Imunidade Inata , Lipopolissacarídeos/metabolismo , Ativação de Macrófagos , Macrófagos/metabolismo , Monócitos/metabolismo , Transdução de Sinais , Transcriptoma , Sistemas de Secreção Tipo IV/genética , Sistemas de Secreção Tipo IV/metabolismo
9.
Virulence ; 12(1): 1610-1628, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34125649

RESUMO

Helicobacter pylori infection is linked to serious gastric-related diseases including gastric cancer. However, current therapies for treating H. pylori infection are challenged by the increased antibiotic resistance of H. pylori. Therefore, it is in an urgent need to identify novel targets for drug development against H. pylori infection. In this study, HP0860 gene from H. pylori predicted to encode a D-glycero-D-manno-heptose-1,7-bisphosphate phosphatase (GmhB) involved in the synthesis of ADP-L-glycero-D-manno-heptose for the assembly of lipopolysaccharide (LPS) in the inner core region was cloned and characterized. We reported HP0860 protein is monomeric and functions as a phosphatase by converting D-glycero-D-manno-heptose-1,7-bisphosphate into D-glycero-D-manno-heptose-1-phosphate with a preference for the ß-anomer over the α-anomer of sugar phosphate substrates. Subsequently, a HP0860 knockout mutant and its complementary mutant were constructed and their phenotypic properties were examined. HP0860 knockout mutant contained both mature and immature forms of LPS and could still induce significant IL-8 secretion after gastric AGS cell infection, suggesting other enzymatic activities in HP0860 knockout mutant might be able to partially compensate for the loss of HP0860 activity. In addition, HP0860 knockout mutant was much more sensitive to antibiotic novobiocin, had decreased adherence abilities, and caused less classic hummingbird phenotype on the infected AGS cells, indicating H. pylori lacking HP0860 is less virulent. Furthermore, the disruption of HP0860 gene altered the sorting of cargo proteins into outer membrane vesicles (OMVs). The above findings confirm the importance of HP0860 in LPS core biosynthesis and shed light on therapeutic intervention against H. pylori infection.


Assuntos
Helicobacter pylori , Heptoses/biossíntese , Monoéster Fosfórico Hidrolases/metabolismo , Virulência , Difosfato de Adenosina , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Técnicas de Inativação de Genes , Infecções por Helicobacter , Helicobacter pylori/enzimologia , Helicobacter pylori/genética , Humanos , Lipopolissacarídeos/biossíntese , Monoéster Fosfórico Hidrolases/genética
10.
Microb Biotechnol ; 14(4): 1797-1808, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34132489

RESUMO

Methanol is a promising feedstock for biomanufacturing of fuels and chemicals. Although efforts have been made to engineer platform microorganisms for methanol bioconversion, the substrate uptake and cell growth rates on methanol are still unsatisfactory, suggesting certain limiting factors remain unsolved. Herein, we analysed the global metabolic regulation changes between an evolved methanol-dependent Corynebacterium glutamicum mutant and its ancestral strain by transcriptome analysis. Many genes involved in central metabolism including glycolysis, amino acid biosynthesis and energy generation were regulated, implying the adaptive laboratory evolution reprogrammed the cellular metabolism for methanol utilization. We then demonstrated that nitrate could serve as a complementary electron acceptor for aerobic methanol metabolism, and the biosynthesis of several amino acids limited methylotrophic growth. Finally, the sedoheptulose bisphosphatase pathway for generating methanol assimilation acceptor was found effective in C. glutamicum. This study identifies limiting factors of methanol metabolism and provides engineering targets for developing superior synthetic methylotrophs.


Assuntos
Corynebacterium glutamicum , Corynebacterium glutamicum/genética , Perfilação da Expressão Gênica , Heptoses , Engenharia Metabólica , Metanol , Nitrogênio
11.
Org Biomol Chem ; 19(22): 4943-4948, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-33988211

RESUMO

d-glycero-d-manno-Heptose-1ß,7-bisphosphate (HBP) is a bacterial metabolite that can induce a TIFA-dependent innate immune response in mammals. It was recently discovered that after HBP enters into the cytoplasm of the host cell, it is transformed into ADP-heptose-7-phosphate, which then leads to ALPK1-TIFA-dependent inflammatory response. In order to provide a molecular tool allowing the discovery of the proteins involved in this novel inflammatory pathway, we designed and synthesized a biotinylated analogue of HBP. This chemical probe displays an anomeric ß-phosphate and a phosphonate at the 7-position, and a d-configured 6-position to which is attached the biotin moiety. To do so, different synthetic strategies were explored and described in this report. Moreover, we demonstrated that the biotinylated version of HBP is still biologically active and can activate the NF-κB pathway in HEK293T cells.


Assuntos
Heptoses
12.
Org Lett ; 23(8): 3216-3220, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33797266

RESUMO

We report a promoter-assisted glycosidation approach for the stereoselective synthesis of the 6-deoxy-ß-d-manno-heptopyranose oligosaccharides. SphosAuNTf2-promoted glycosidation of 6-deoxy-d-manno-heptopyranosyl o-hexynylbenzoate with common alcohols afforded a range of 6-deoxy-d-manno-heptosides with good to excellent ß-selectivities. The counterion and the ligand of SPhosAuNTf2 were found to have a dramatic effect on the formation of the 1,2-cis-ß-linked 6-deoxy-d-manno-heptosides. This approach was effectively applied to the stereocontrolled synthesis of the 6-deoxy-ß-d-manno-heptopyranose oligosaccharides relevant to Burkholderia pseudomallei and Burkholderia mallei.


Assuntos
Heptoses/síntese química , Oligossacarídeos/síntese química , Burkholderia/química , Glicosilação , Heptoses/química , Estrutura Molecular , Oligossacarídeos/química
13.
Biochemistry ; 60(19): 1552-1563, 2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-33900734

RESUMO

Campylobacter jejuni is the leading cause of food poisoning in the United States and Europe. The exterior cell surface of C. jejuni is coated with a capsular polysaccharide (CPS) that is essential for the maintenance and integrity of the bacterial cell wall and evasion of the host immune response. The identity and sequences of the monosaccharide components of the CPS are quite variable and dependent on the specific strain of C. jejuni. It is currently thought that the immediate precursor for the multiple variations found in the heptose moieties of the C. jejuni CPS is GDP-d-glycero-α-d-manno-heptose. In C. jejuni NCTC 11168, the heptose moiety is d-glycero-l-gluco-heptose. It has previously been shown that Cj1427 catalyzes the oxidation of GDP-d-glycero-α-d-manno-heptose to GDP-d-glycero-4-keto-α-d-lyxo-heptose using α-ketoglutarate as a cosubstrate. Cj1430 was now demonstrated to catalyze the double epimerization of this product at C3 and C5 to form GDP-d-glycero-4-keto-ß-l-xylo-heptose. Cj1428 subsequently catalyzes the stereospecific reduction of this GDP-linked heptose by NADPH to form GDP-d-glycero-ß-l-gluco-heptose. The three-dimensional crystal structure of Cj1430 was determined to a resolution of 1.85 Å in the presence of bound GDP-d-glycero-ß-l-gluco-heptose, a product analogue. The structure shows that it belongs to the cupin superfamily. The three-dimensional crystal structure of Cj1428 was solved in the presence of NADPH to a resolution of 1.50 Å. Its fold places it into the short-chain dehydrogenase/reductase superfamily. Typically, members in this family display a characteristic signature sequence of YXXXK, with the conserved tyrosine serving a key role in catalysis. In Cj1428, this residue is a phenylalanine.


Assuntos
Campylobacter jejuni/metabolismo , Heptoses/biossíntese , Proteínas de Bactérias/química , Campylobacter jejuni/patogenicidade , Guanosina Difosfato/metabolismo , Heptoses/química , Heptoses/metabolismo , Ácidos Cetoglutáricos/metabolismo , Monossacarídeos/metabolismo , Oxirredutases/metabolismo , Polissacarídeos/metabolismo , Polissacarídeos Bacterianos/biossíntese , Polissacarídeos Bacterianos/metabolismo
14.
Nat Prod Rep ; 38(10): 1887-1909, 2021 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-33704304

RESUMO

Covering: up to 2020Glycosylated natural products hold great potential as drugs for the treatment of human and animal diseases. Heptoses, known as seven-carbon-chain-containing sugars, are a group of saccharides that are rarely observed in natural products. Based on the structures of the heptoses, the heptose-containing natural products can be divided into four groups, characterized by heptofuranose, highly-reduced heptopyranose, D-heptopyranose, and L-heptopyranose. Many of them possess remarkable biological properties, including antibacterial, antifungal, antitumor, and pain relief activities, thereby attracting great interest in biosynthesis and chemical synthesis studies to understand their construction mechanisms and structure-activity relationships. In this review, we summarize the structural properties, biological activities, and recent progress in the biosynthesis of bacterial natural products featuring seven-carbon-chain-containing sugars. The biosynthetic origins of the heptose moieties are emphasized.


Assuntos
Bactérias/metabolismo , Produtos Biológicos/metabolismo , Heptoses/biossíntese , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/farmacologia , Heptoses/química , Heptoses/isolamento & purificação , Heptoses/farmacologia
15.
J Biol Chem ; 296: 100352, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33524389

RESUMO

Many bacteria produce polysaccharide-based capsules that protect them from environmental insults and play a role in virulence, host invasion, and other functions. Understanding how the polysaccharide components are synthesized could provide new means to combat bacterial infections. We have previously characterized two pairs of homologous enzymes involved in the biosynthesis of capsular sugar precursors GDP-6-deoxy-D-altro-heptose and GDP-6-OMe-L-gluco-heptose in Campylobacter jejuni. However, the substrate specificity and mechanism of action of these enzymes-C3 and/or C5 epimerases DdahB and MlghB and C4 reductases DdahC and MlghC-are unknown. Here, we demonstrate that these enzymes are highly specific for heptose substrates, using mannose substrates inefficiently with the exception of MlghB. We show that DdahB and MlghB feature a jellyroll fold typical of cupins, which possess a range of activities including epimerizations, GDP occupying a similar position as in cupins. DdahC and MlghC contain a Rossman fold, a catalytic triad, and a small C-terminal domain typical of short-chain dehydratase reductase enzymes. Integrating structural information with site-directed mutagenesis allowed us to identify features unique to each enzyme and provide mechanistic insight. In the epimerases, mutagenesis of H67, D173, N121, Y134, and Y132 suggested the presence of alternative catalytic residues. We showed that the reductases could reduce GDP-4-keto-6-deoxy-mannulose without prior epimerization although DdahC preferred the pre-epimerized substrate and identified T110 and H180 as important for substrate specificity and catalytic efficacy. This information can be exploited to identify inhibitors for therapeutic applications or to tailor these enzymes to synthesize novel sugars useful as glycobiology tools.


Assuntos
Proteínas de Bactérias/metabolismo , Campylobacter jejuni/metabolismo , Heptoses/metabolismo , Oxirredutases/metabolismo , Racemases e Epimerases/metabolismo , Proteínas de Bactérias/química , Infecções por Campylobacter/microbiologia , Campylobacter jejuni/química , Heptoses/química , Humanos , Oxirredutases/química , Conformação Proteica , Racemases e Epimerases/química , Especificidade por Substrato
16.
J Org Chem ; 86(3): 2184-2199, 2021 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-33449680

RESUMO

Pathogen-associated molecular patterns activate the immune system via pattern recognition receptors. Recently, newly discovered pathogen-associated molecular patterns, d-glycero-ß-d-mannoheptose phosphate and d-glycero-ß-d-mannoheptose 1,7-biphosphate, were shown to induce a TRAF-interacting protein with a forkhead-associated domain-dependent immune response in human embryonic kidney cells and colonic epithelial cells. Concurrently, ADP-heptose was shown to bind α-kinase 1 and activate TIFA via phosphorylation leading to an immune cascade to ultimately activate NF-κB. These pathogen-associated molecular patterns have raised interest in the pharmaceutical industry for their potential use as immunomodulators. However, little is understood about the host cell uptake of d-glycero-ß-d-mannoheptose phosphate, d-glycero-ß-d-mannoheptose 1,7-biphosphate, and ADP-heptose in vivo and derivatives of these molecules are needed to interrogate this. In this regard, herein we describe 7-O-modifications of d-glycero-ß-d-mannoheptose phosphate to produce molecular probes toward the development of a useful toolbox for biologists. A convergent strategy that involves introduction of a substituent at O-7 before alkene oxidation was investigated and proved successful in the generation of a range of molecular probes.


Assuntos
Heptoses , Fosfatos , Humanos , Fatores Imunológicos , Fosforilação
17.
Cell Mol Life Sci ; 78(1): 17-29, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32591860

RESUMO

The innate immune response constitutes the first line of defense against pathogens. It involves the recognition of pathogen-associated molecular patterns (PAMPs) by pathogen recognition receptors (PRRs), the production of inflammatory cytokines and the recruitment of immune cells to infection sites. Recently, ADP-heptose, a soluble intermediate of the lipopolysaccharide biosynthetic pathway in Gram-negative bacteria, has been identified by several research groups as a PAMP. Here, we recapitulate the evidence that led to this identification and discuss the controversy over the immunogenic properties of heptose 1,7-bisphosphate (HBP), another bacterial heptose previously defined as an activator of innate immunity. Then, we describe the mechanism of ADP-heptose sensing by alpha-protein kinase 1 (ALPK1) and its downstream signaling pathway that involves the proteins TIFA and TRAF6 and induces the activation of NF-κB and the secretion of inflammatory cytokines. Finally, we discuss possible delivery mechanisms of ADP-heptose in cells during infection, and propose new lines of thinking to further explore the roles of the ADP-heptose/ALPK1/TIFA axis in infections and its potential implication in the control of intestinal homeostasis.


Assuntos
Heptoses/metabolismo , Padrões Moleculares Associados a Patógenos/metabolismo , Proteínas Quinases/metabolismo , Citocinas/metabolismo , Bactérias Gram-Negativas/imunologia , Bactérias Gram-Negativas/metabolismo , Humanos , Imunidade Inata , Lipopolissacarídeos/biossíntese , Lipopolissacarídeos/química , NF-kappa B/metabolismo , Transdução de Sinais
18.
Nutrients ; 14(1)2021 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-35011030

RESUMO

Beginning at 16 weeks of age and continuing for 44 weeks, male C57BL/6J were fed either a control (CON) diet; a high-fat (HF) diet (60% unsaturated); or the HF diet containing an extract of unripe avocados (AvX) enriched in the 7-carbon sugar mannoheptulose (MH), designed to act as a glycolytic inhibitor (HF + MH). Compared to the CON diet, mice on the HF diet exhibited higher body weights; body fat; blood lipids; and leptin with reduced adiponectin levels, insulin sensitivity, VO2max, and falls from a rotarod. Mice on the HF + MH diet were completely protected against these changes in the absence of significant diet effects on food intake. Compared to the CON diet, oxidative stress was also increased by the HF diet indicated by higher levels of total reactive oxygen species, superoxide, and peroxynitrite measured in liver samples by electron paramagnetic resonance spectroscopy, whereas the HF + MH diet attenuated these changes. Compared to the CON, the HF diet increased signaling in the mechanistic target of the rapamycin (mTOR) pathway, and the addition of the MH-enriched AvX to this diet attenuated these changes. Beyond generating further interest in the health benefits of avocados, these results draw further new attention to the effects of this rare sugar, MH, as a botanical intervention for preventing obesity.


Assuntos
Dieta Hiperlipídica/efeitos adversos , Suplementos Nutricionais , Heptoses/administração & dosagem , Obesidade/etiologia , Obesidade/prevenção & controle , Persea/química , Fitoterapia , Extratos Vegetais/administração & dosagem , Animais , Heptoses/análise , Heptoses/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/análise , Extratos Vegetais/farmacologia , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo
19.
Org Lett ; 22(22): 8780-8785, 2020 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-33119312

RESUMO

Helicobacter pylori, the most common cause of chronic gastritis, peptic ulcers, and gastric cancers, infects around half of the world's population. Although the drawbacks of antibiotic-based combination therapy are emerging, no effective vaccine is available to prevent H. pylori infections. Here, we describe the total synthesis of the unique α-(1→3)-linked tri-d-glycero-d-manno-heptose antigen from the lipopolysaccharide of H. pylori serogroups O3 and O6 and strains MO19, D2, D4, and D5 based on de novo synthesis of the differentially protected d-glycero-d-manno-heptosyl building blocks. Immunization of mice with the semisynthetic glycoconjugate elicited a very robust T-cell-dependent antigen-specific immune response, resulting in very high titers of IgG1 and IgG2b protective antibody isotypes. The postimmune sera recognized H. pylori NCTC 11637 and bound strongly to the surface of the intact bacteria.


Assuntos
Helicobacter pylori/imunologia , Heptoses/síntese química , Lipopolissacarídeos/química , Animais , Glicoconjugados/química , Helicobacter pylori/química , Heptoses/imunologia , Camundongos , Estrutura Molecular , Vacinas/imunologia
20.
Org Lett ; 22(20): 8018-8022, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-32991182

RESUMO

Synthesis of bacterial cell surface l-glycero-d-manno-heptose (l,d-Hep)- and d-glycero-d-manno-heptose (d,d-Hep)-containing higher carbon sugars is a challenging task. Here, we report a convenient and efficient approach for the synthesis of the l,d-Hep and d,d-Hep building blocks. Using l-lyxose and d-ribose as starting materials, this approach features diastereoselective Mukaiyama-type aldol reactions as the key steps. On the basis of the synthetic l,d-Hep and d,d-Hep building blocks, we achieved the first stereoselective synthesis of the unique α-l,d-Hep-(1→3)-α-d,d-Hep-(1→5)-α-Kdo core trisaccharide of the lipopolysaccharide of Vibrio parahemolyticus O2.


Assuntos
Heptoses/síntese química , Lipopolissacarídeos/síntese química , Trissacarídeos/síntese química , Vibrio/química , Heptoses/química , Lipopolissacarídeos/química , Estrutura Molecular , Trissacarídeos/química
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