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1.
J Agric Food Chem ; 67(31): 8573-8580, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31293156

RESUMO

Glycosylation endows both natural and synthetic small molecules with modulated physicochemical and biological properties. Plant and bacterial glycosyltransferases capable of decorating various privileged scaffolds have been extensively studied, but those from kingdom Fungi still remain underexploited. Here, we use a combination of genome mining and heterologous expression techniques to identify four novel glycosyltransferase-methyltransferase (GT-MT) functional modules from Hypocreales fungi. These GT-MT modules display decent substrate promiscuity and regiospecificity, methylglucosylating a panel of natural products such as flavonoids, stilbenoids, anthraquinones, and benzenediol lactones. Native GT-MT modules can be split up and regrouped into hybrid modules with similar or even improved efficacy as compared with native pairs. Methylglucosylation of kaempferol considerably improves its insecticidal activity against the larvae of oriental armyworm Mythimna separata (Walker). Our work provides a set of efficient biocatalysts for the combinatorial biosynthesis of small molecule glycosides that may have significant importance to the pharmaceutical, agricultural, and food industries.


Assuntos
Proteínas Fúngicas/química , Glicosiltransferases/química , Hypocreales/enzimologia , Metiltransferases/química , Fenóis/química , Animais , Biocatálise , Cristalografia por Raios X , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glicosilação , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Hypocreales/genética , Inseticidas/química , Inseticidas/farmacologia , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Mariposas/efeitos dos fármacos , Fenóis/farmacologia , Especificidade por Substrato
2.
J Agric Food Chem ; 67(23): 6541-6550, 2019 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-31125230

RESUMO

The enzymatic hydrolysis of glycosidically bound volatiles (GBVs) plays an important role in tea aroma formation during the tea manufacturing process. However, during the enzyme-active manufacturing process of oolong tea, most GBVs showed no reduction, while ( Z)-3-hexenyl-ß-glucopyranoside significantly enhanced at the turnover stage. This study aimed to determine the reason for this increase in ( Z)-3-hexenyl-ß-glucopyranoside. Continuous wounding stress in the turnover stage did not enhance the expression level of glycosyltransferase 1 ( CsGT1), while it induced a significant increase in the ( Z)-3-hexenol content ( p ≤ 0.05). Furthermore, observing the cell structures of tea leaves exposed to continuous wounding and subcellular localizations of CsGTs suggested that the interaction of ( Z)-3-hexenol (substrate) and CsGT1 (enzyme) was available. In conclusion, both continuous wounding and subcellular localizations led to a ( Z)-3-hexenyl-ß-glucopyranoside enhancement mechanism during the oolong tea's turnover stage. These results advance our understanding of GBV formation during the tea manufacturing process and their relationship with the stress from the tea manufacturing process. In addition, the information will help us further evaluate contribution of GBVs to enzymatic formation of oolong tea aroma compounds.


Assuntos
Camellia sinensis/química , Manipulação de Alimentos/métodos , Glucosídeos/análise , Camellia sinensis/enzimologia , Camellia sinensis/genética , Camellia sinensis/fisiologia , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Folhas de Planta/química , Folhas de Planta/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estresse Fisiológico , Compostos Orgânicos Voláteis/análise
3.
Org Lett ; 21(9): 3252-3256, 2019 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-30977375

RESUMO

A rare fungal hopane-type triterpenoid glycoside fumihopaside A was identified by genome mining in combination with HPLC-MS/MS in Aspergillus fumigatus. Combining genetic deletions in A. fumigatus with heterologous reconstitutions in Aspergillus nidulans of the afum gene cluster, we identified one fungal squalene hopane cyclase AfumA charging the cyclization of the hopene skeleton, one cytochrome P450, and one UDP-glycosyltransferase. Bioassays indicated that fumihopaside A plays important roles in protecting A. fumigatus against heat or ultraviolet stress.


Assuntos
Aspergillus fumigatus/metabolismo , Glicosídeos/biossíntese , Triterpenos/metabolismo , Aspergillus fumigatus/genética , Aspergillus nidulans/genética , Ciclização , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Genoma Fúngico , Glicosídeos/química , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Família Multigênica , Metabolismo Secundário , Triterpenos/química
4.
Nat Commun ; 10(1): 1669, 2019 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-30971696

RESUMO

Fungal cell wall synthesis is achieved by a balance of glycosyltransferase, hydrolase and transglycosylase activities. Transglycosylases strengthen the cell wall by forming a rigid network of crosslinks through mechanisms that remain to be explored. Here we study the function of the Aspergillus fumigatus family of five Crh transglycosylases. Although crh genes are dispensable for cell viability, simultaneous deletion of all genes renders cells sensitive to cell wall interfering compounds. In vitro biochemical assays and localisation studies demonstrate that this family of enzymes functions redundantly as transglycosylases for both chitin-glucan and chitin-chitin cell wall crosslinks. To understand the molecular basis of this acceptor promiscuity, we solved the crystal structure of A. fumigatus Crh5 (AfCrh5) in complex with a chitooligosaccharide at the resolution of 2.8 Å, revealing an extensive elongated binding cleft for the donor (-4 to -1) substrate and a short acceptor (+1 to +2) binding site. Together with mutagenesis, the structure suggests a "hydrolysis product assisted" molecular mechanism favouring transglycosylation over hydrolysis.


Assuntos
Aspergillus fumigatus/metabolismo , Proteínas Fúngicas/metabolismo , Glicosiltransferases/metabolismo , Sítios de Ligação/genética , Parede Celular/metabolismo , Quitina/metabolismo , Cristalografia por Raios X , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Técnicas de Silenciamento de Genes , Glicosiltransferases/química , Glicosiltransferases/genética , Mutagênese Sítio-Dirigida , Domínios Proteicos/genética , Especificidade por Substrato , beta-Glucanas/metabolismo
5.
Nat Commun ; 10(1): 1799, 2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30996301

RESUMO

Chemoenzymatic modification of cell-surface glycan structures has emerged as a complementary approach to metabolic oligosaccharide engineering. Here, we identify Pasteurella multocida α2-3-sialyltransferase M144D mutant, Photobacterium damsela α2-6-sialyltransferase, and Helicobacter mustelae α1-2-fucosyltransferase, as efficient tools for live-cell glycan modification. Combining these enzymes with Helicobacter pylori α1-3-fucosyltransferase, we develop a host-cell-based assay to probe glycan-mediated influenza A virus (IAV) infection including wild-type and mutant strains of H1N1 and H3N2 subtypes. At high NeuAcα2-6-Gal levels, the IAV-induced host-cell death is positively correlated with haemagglutinin (HA) binding affinity to NeuAcα2-6-Gal. Remarkably, an increment of host-cell-surface sialyl Lewis X (sLeX) exacerbates the killing by several wild-type IAV strains and a previously engineered mutant HK68-MTA. Structural alignment of HAs from HK68 and HK68-MTA suggests formation of a putative hydrogen bond between Trp222 of HA-HK68-MTA and the C-4 hydroxyl group of the α1-3-linked fucose of sLeX, which may account for the enhanced host cell killing of that mutant.


Assuntos
Proteínas de Bactérias/metabolismo , Glicosiltransferases/metabolismo , Hemaglutininas/imunologia , Interações Hospedeiro-Patógeno/imunologia , Influenza Humana/imunologia , Oligossacarídeos/metabolismo , Animais , Proteínas de Bactérias/genética , Bioensaio/métodos , Células CHO , Cricetulus , Cães , Glicosiltransferases/genética , Voluntários Saudáveis , Helicobacter mustelae/genética , Helicobacter mustelae/metabolismo , Hemaglutininas/metabolismo , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/imunologia , Influenza Humana/virologia , Microscopia Intravital/métodos , Luciferases Bacterianas/genética , Luciferases Bacterianas/metabolismo , Pulmão/patologia , Células Madin Darby de Rim Canino , Engenharia Metabólica/métodos , Microscopia de Fluorescência/métodos , Imagem Molecular/métodos , Oligossacarídeos/imunologia , Pasteurella multocida/genética , Pasteurella multocida/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Coloração e Rotulagem/métodos
6.
Org Lett ; 21(7): 2241-2245, 2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-30848604

RESUMO

A highly regio-specific and donor-promiscuous 3- O-glycosyltransferase, Sb3GT1 (UGT78B4), was discovered from Scutellaria baicalensis. Sb3GT1 could accept five sugar donors (UDP-Glc/-Gal/-GlcNAc/-Xyl/-Ara) to catalyze 3- O-glycosylation of 17 flavonols, and the conversion rates could be >98%. Five new glycosides were obtained by scaled-up enzymatic catalysis. Molecular modeling and site-directed mutagenesis revealed that G15 and P187 were critical catalytic residues for the donor promiscuity. Sb3GT1 could be a promising catalyst to increase structural diversity of flavonoid 3- O-glycosides.


Assuntos
Flavonoides/metabolismo , Glicosídeos/química , Glicosiltransferases/metabolismo , Scutellaria baicalensis/química , Flavonoides/química , Flavonoides/isolamento & purificação , Glicosilação , Glicosiltransferases/química , Glicosiltransferases/isolamento & purificação , Modelos Moleculares , Estrutura Molecular
7.
Methods Mol Biol ; 1954: 151-159, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30864130

RESUMO

The incorporation of fluorescent tags into synthetic acceptor molecules for in vitro biochemical assays allows quick and easy detection of enzyme activity. Reaction products can be separated via thin-layer chromatography and visualized under UV light for rapid detection of reaction progress. Subsequent structural analysis of these reaction products through the use of NMR spectroscopy and mass spectrometry allows for complete functional characterization of enzyme activity. Here we describe an application of this technique which was previously used to functionally characterize a dual-domain glycosyltransferase enzyme, KpsC, involved in capsular polysaccharide biosynthesis in Escherichia coli.


Assuntos
Ensaios Enzimáticos/métodos , Escherichia coli/enzimologia , Corantes Fluorescentes/metabolismo , Glicosiltransferases/metabolismo , Cromatografia em Camada Delgada/métodos , Escherichia coli/metabolismo , Corantes Fluorescentes/análise , Glicosiltransferases/análise , Espectrometria de Massas/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , Especificidade por Substrato
8.
Methods Mol Biol ; 1954: 215-235, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30864135

RESUMO

Glycans play many important roles in bacterial biology and the complexity of the glycan structures requires biochemical assays in place to help characterize the biosynthetic pathways. Our focus has been on the use of enzymes from pathogens which make molecular mimics of host glycans. We have been examining glycosyltransferases that make strategic linkages in biologically active glycans which can be also exploited for potential therapeutic glycoconjugate synthesis. This chapter will provide details on assays for a variety of bacterial glycosyltransferases that we and others have used for the characterization of pathogen glycoconjugate biosynthetic pathways, and for the in vitro synthesis of human-like glycans produced by bacterial pathogens. The methods presented here should enable other assays to be developed for new pathway characterization.


Assuntos
Bactérias/enzimologia , Compostos de Boro/metabolismo , Ensaios Enzimáticos/métodos , Corantes Fluorescentes/metabolismo , Glicosiltransferases/metabolismo , Polissacarídeos Bacterianos/metabolismo , Bactérias/química , Bactérias/metabolismo , Vias Biossintéticas , Compostos de Boro/análise , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia em Camada Delgada/métodos , Corantes Fluorescentes/análise , Oxirredução , Polissacarídeos Bacterianos/análise , Proteínas Recombinantes/metabolismo
9.
Methods Mol Biol ; 1954: 237-243, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30864136

RESUMO

The glycosyltransferases (GTs) are an important subclass of enzymes that catalyze the biosynthesis of glycosidic bonds in oligosaccharides, polysaccharides and glycoconjugates by transferring a sugar residue from a donor substrate to an acceptor substrate. The membrane-associated GTs play a vital role in the biosynthesis of bacterial cell-wall polysaccharides. Characterization and quantification of GT activities is important for studies of biosynthesis of polysaccharides, drug target development, and production of bacterial products. In this chapter, colorimetric assays for the measurement of GT activities will be presented. Assays for GTs acting on monosaccharide-derivatives are based on the cleavage of unreacted glycosyl-p-nitrophenol acceptors followed by detection of p-nitrophenolate. GT reactions coupled with phosphatases and detection of inorganic phosphate are suitable for most GTs. These assays permit convenient quantification of GT activities and kinetics without the use of radioactive sugars.


Assuntos
Proteínas de Bactérias/metabolismo , Colorimetria/métodos , Ensaios Enzimáticos/métodos , Glicosiltransferases/metabolismo , Neisseria meningitidis/enzimologia , Proteínas de Bactérias/análise , Glicosiltransferases/análise , Humanos , Cinética , Infecções Meningocócicas/microbiologia , Monossacarídeos/metabolismo , Neisseria meningitidis/química , Nitrofenóis/análise , Nitrofenóis/metabolismo , Especificidade por Substrato
10.
Methods Mol Biol ; 1954: 245-253, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30864137

RESUMO

In vitro assays using fluorescently tagged sugar residues can facilitate the characterization of glycosyltransferase function. Here we describe the use of in vitro assays to characterize the three glycosyltransferase modules of the protein designated WbbB from Klebsiella pneumoniae O12. This protein combines key activities necessary to synthesize the O antigenic polysaccharide portion of lipopolysaccharide. The specificities of the three glycosyltransferases were investigated in vitro, using purified proteins, the activated donor sugars (dTDP-Rha, UDP-GlcNAc and CMP-ß-Kdo) and synthetic acceptors terminating in either α1,3-linked Rha or ß1,4-linked GlcNAc. The reaction products were verified by mass spectrometry and nuclear magnetic resonance methods.


Assuntos
Proteínas de Bactérias/metabolismo , Glicosiltransferases/metabolismo , Klebsiella pneumoniae/enzimologia , Proteínas de Bactérias/química , Cromatografia em Camada Delgada/métodos , Corantes Fluorescentes/análise , Corantes Fluorescentes/metabolismo , Glicosiltransferases/química , Humanos , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/química , Klebsiella pneumoniae/metabolismo , Espectrometria de Massas/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , Antígenos O/metabolismo , Domínios Proteicos
11.
Methods Mol Biol ; 1954: 255-268, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30864138

RESUMO

The donor substrates for the biosynthesis of bacterial polysaccharides include UDP-Glc/Gal and UDP-GlcNAc/GalNAc. The conversion of these nucleotide sugars is catalyzed by 4-epimerases. The wbpP gene of Pseudomonas aeruginosa encodes a 4-epimerase that has a preference for UDP-GlcNAc/GalNAc as substrates. Other 4-epimerases have broad specificities or preference for UDP-Glc/Gal. We have developed coupled assays where the 4-epimerase product is used as a donor substrate for glycosyltransferases that are highly specific for the nucleotide sugar structure. We describe here a method for the study of substrate specificity of WbpP, using coupled assays employing four different glycosyltransferases. These protocols can be applied to the identification and characterization of novel 4-epimerases and to determine their substrate specificities.


Assuntos
Ensaios Enzimáticos/métodos , Glicosiltransferases/metabolismo , Pseudomonas aeruginosa/enzimologia , Racemases e Epimerases/metabolismo , Polissacarídeos Bacterianos/metabolismo , Pseudomonas aeruginosa/metabolismo , Especificidade por Substrato
12.
Methods Mol Biol ; 1954: 279-296, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30864140

RESUMO

Antimicrobial peptides (AMPs) are novel agents for therapeutic application for their inherent broad spectrum of activities against bacteria, fungi, and viruses, as well as anti-inflammatory, anticancerous, and immunomodulatory activities. This chapter presents an enzymatic method to generate glycovariants of one such antimicrobial peptide, namely enterocin 96, using a bacterial protein O- and S-glycosyltransferase, in vitro.


Assuntos
Antibacterianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas de Bactérias/metabolismo , Enterococcus faecalis/metabolismo , Glicosiltransferases/metabolismo , Sequência de Aminoácidos , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Proteínas de Bactérias/genética , Cromatografia Líquida de Alta Pressão/métodos , Clonagem Molecular/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Enterococcus faecalis/química , Enterococcus faecalis/genética , Ensaios Enzimáticos/métodos , Escherichia coli/genética , Genes Bacterianos , Glicosilação , Glicosiltransferases/genética , Espectrometria de Massas/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
13.
Malar J ; 18(1): 62, 2019 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-30845961

RESUMO

BACKGROUND: UDP-glycosyltransferase (UGT) is an important biotransformation superfamily of enzymes. They catalyze the transfer of glycosyl residues from activated nucleotide sugars to acceptor hydrophobic molecules, and function in several physiological processes, including detoxification, olfaction, cuticle formation, pigmentation. The diversity, classification, scaffold location, characteristics, phylogenetics, and evolution of the superfamily of genes at whole genome level, and their association and mutations associated with pyrethroid resistance are still little known. METHODS: The present study identified UGT genes in Anopheles sinensis genome, classified UGT genes in An. sinensis, Anopheles gambiae, Aedes aegypti and Drosophila melanogaster genomes, and analysed the scaffold location, characteristics, phylogenetics, and evolution of An. sinensis UGT genes using bioinformatics methods. The present study also identified the UGTs associated with pyrethroid resistance using three field pyrethroid-resistant populations with RNA-seq and RT-qPCR, and the mutations associated with pyrethroid resistance with genome re-sequencing in An. sinensis. RESULTS: There are 30 putative UGTs in An. sinensis genome, which are classified into 12 families (UGT301, UGT302, UGT306, UGT308, UGT309, UGT310, UGT313, UGT314, UGT315, UGT36, UGT49, UGT50) and further into 23 sub-families. The UGT308 is significantly expanded in gene number compared with other families. A total of 119 UGTs from An. sinensis, An. gambiae, Aedes aegypti and Drosophila melanogaster genomes are classified into 19 families, of which seven are specific for three mosquito species and seven are specific for Drosophila melanogaster. The UGT308 and UGT302 are proposed to main families involved in pyrethroid resistance. The AsUGT308D3 is proposed to be the essential UGT gene for the participation in biotransformation in pyrethroid detoxification process, which is possibly regulated by eight SNPs in its 3' flanking region. The UGT302A3 is also associated with pyrethroid resistance, and four amino acid mutations in its coding sequences might enhance its catalytic activity and further result in higher insecticide resistance. CONCLUSIONS: This study provides the diversity, phylogenetics and evolution of UGT genes, and potential UGT members and mutations involved in pyrethroid resistance in An. sinensis, and lays an important basis for the better understanding and further research on UGT function in defense against insecticide stress.


Assuntos
Anopheles/efeitos dos fármacos , Anopheles/enzimologia , Glicosiltransferases/genética , Resistência a Inseticidas , Inseticidas/farmacologia , Proteínas Mutantes/genética , Piretrinas/farmacologia , Aedes/enzimologia , Aedes/genética , Animais , Anopheles/genética , Biologia Computacional , Drosophila/enzimologia , Drosophila/genética , Feminino , Perfilação da Expressão Gênica , Glicosiltransferases/metabolismo , Proteínas Mutantes/metabolismo , Mutação , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de RNA
14.
Phytochemistry ; 162: 141-147, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30897351

RESUMO

In the metabolic glycosylation grid of steviol glycosides, UGT76G1 was shown to catalyze at least eight different glucosylation steps, including the formation of rebaudioside B (Reb B) and rebaudioside A (Reb A) (Olsson et al., 2016). In this study, the accumulation of steviolbioside, Reb B, stevioside (ST) and Reb A in more than 140 samples of stevia leaves collected from different regions in China were analyzed by high-performance liquid chromatography (HPLC), and five genotypes, 'N01-N05', with significantly different levels of the abovementioned glycosides were discovered. Mutations in the UGT76G1 gene cloned from cDNAs from these five genotypes were identified, and the functions of the recombinant UGT76G1 variants were ascertained by adding steviolbioside and ST substrates. In addition, homology modeling and molecular docking were used to elucidate the functional differences between variants and UGT76G1. Comparing the sequences of the five variants 'N01-N05' with UGT76G1 (AY345974.1) revealed that base substitutions were not observed in 'N01'. By contrast, 'N02' exhibited 9 single nucleotide polymorphisms (SNPs) and 9 associated amino acid substitutions or insertions with notable variations in the protein structure; however, an enzyme assay showed similar functionalities between the variant and UGT76G1. In 'N03', 49 SNPs and 29 associated amino acid substitutions or insertions were identified and shown to induce significant variations in the protein structure, especially in the binding pocket, resulting in the lack of functionality of this variant in the enzyme assay. These results were in agreement with the docking profiles. Moreover, a nonsense mutation of p.1090T > G in 'N04' and an insertion of a 68 base fragment in 'N05' were found, and both produced a premature protein without any catalytic activity. Therefore, UGT76G1, which is vital to the content of main steviol glycosides, should be a key gene marker for the molecular breeding of Stevia rebaudiana. Our investigations also revealed the location and orientation of active groups of the receptors and donors in the UGT76G1 enzyme, which play key roles in determining whether the enzyme has any enzymatic activity.


Assuntos
Diterpenos de Caurano/metabolismo , Glucosídeos/metabolismo , Glicosiltransferases/genética , Mutação , Stevia/metabolismo , Difosfato de Uridina/metabolismo , Biocatálise , Clonagem Molecular , Glicosiltransferases/química , Glicosiltransferases/metabolismo , Modelos Moleculares , Conformação Proteica , Stevia/enzimologia , Stevia/genética
15.
Int J Mol Sci ; 20(5)2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30823656

RESUMO

Uridine diphosphate glycosyltransferases (UGTs) are multifunctional detoxification enzymes, which are involved in metabolizing various chemicals and contribute to the development of insecticide resistance. However, the possible roles of UGTs in chlorantraniliprole resistance in Chilo suppressalis have rarely been studied in detail. Based on genome data, 24 UGT genes in C. suppressalis belonging to 11 families were identified, which were designated by the UGT nomenclature committee. Synergism assay data suggested that UGTs are potentially involved in chlorantraniliprole resistance in C. suppressalis. CsUGT40AL1 and CsUGT33AG3 were significantly overexpressed in the chlorantraniliprole resistant strain (12.36- and 5.34-fold, respectively). The two UGTs were highly expressed in the larval Malpighian tubules, fat body, and midgut; however, expression was lowest in the head. Injection of individual dsRNAs reduced the expression of the two target genes (by 69.34% and 48.74%, respectively) and caused significant higher larval mortality (81.33% and 54.67%, respectively). Overexpression of CsUGT40AL1 and CsUGT33AG3 was potentially involved in chlorantraniliprole resistance in C. suppressalis, as confirmed by the RNAi assay. Our findings suggest that overexpression of UGTs may contribute to chlorantraniliprole resistance in C. suppressalis.


Assuntos
Glicosiltransferases/genética , Proteínas de Insetos/genética , Resistência a Inseticidas , Lepidópteros/genética , Animais , Glicosiltransferases/metabolismo , Proteínas de Insetos/metabolismo , Inseticidas/toxicidade , Lepidópteros/efeitos dos fármacos , Especificidade de Órgãos , ortoaminobenzoatos/toxicidade
16.
Plant Mol Biol ; 100(1-2): 181-197, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30868545

RESUMO

KEY MESSAGE: The knowledge of substrate specificity of XET enzymes is important for the general understanding of metabolic pathways to challenge the established notion that these enzymes operate uniquely on cellulose-xyloglucan networks. Xyloglucan xyloglucosyl transferases (XETs) (EC 2.4.1.207) play a central role in loosening and re-arranging the cellulose-xyloglucan network, which is assumed to be the primary load-bearing structural component of plant cell walls. The sequence of mature TmXET6.3 from Tropaeolum majus (280 residues) was deduced by the nucleotide sequence analysis of complete cDNA by Rapid Amplification of cDNA Ends, based on tryptic and chymotryptic peptide sequences. Partly purified TmXET6.3, expressed in Pichia occurred in N-glycosylated and unglycosylated forms. The quantification of hetero-transglycosylation activities of TmXET6.3 revealed that (1,3;1,4)-, (1,6)- and (1,4)-ß-D-glucooligosaccharides were the preferred acceptor substrates, while (1,4)-ß-D-xylooligosaccharides, and arabinoxylo- and glucomanno-oligosaccharides were less preferred. The 3D model of TmXET6.3, and bioinformatics analyses of identified and putative plant xyloglucan endotransglycosylases (XETs)/hydrolases (XEHs) of the GH16 family revealed that H94, A104, Q108, K234 and K237 were the key residues that underpinned the acceptor substrate specificity of TmXET6.3. Compared to the wild-type enzyme, the single Q108R and K237T, and double-K234T/K237T and triple-H94Q/A104D/Q108R variants exhibited enhanced hetero-transglycosylation activities with xyloglucan and (1,4)-ß-D-glucooligosaccharides, while those with (1,3;1,4)- and (1,6)-ß-D-glucooligosaccharides were suppressed; the incorporation of xyloglucan to (1,4)-ß-D-glucooligosaccharides by the H94Q variant was influenced most extensively. Structural and biochemical data of non-specific TmXET6.3 presented here extend the classic XET reaction mechanism by which these enzymes operate in plant cell walls. The evaluations of TmXET6.3 transglycosylation activities and the incidence of investigated residues in other members of the GH16 family suggest that a broad acceptor substrate specificity in plant XET enzymes could be more widespread than previously anticipated.


Assuntos
Glicosiltransferases/metabolismo , Proteínas de Plantas/metabolismo , Engenharia de Proteínas , Sementes/enzimologia , Tropaeolum/enzimologia , Sequência de Aminoácidos , Sequência de Bases , DNA Complementar/genética , Germinação , Glicosilação , Glicosiltransferases/química , Modelos Moleculares , Petroselinum/enzimologia , Filogenia , Proteínas de Plantas/química , Homologia Estrutural de Proteína , Especificidade por Substrato
17.
Int J Biol Macromol ; 130: 536-544, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30802520

RESUMO

Klebsiella pneumoniae strain KK207-2 was isolated in 2010 from a bloodstream infection of an inpatient at an Italian hospital. It was previously found to produce the KPC-2 carbapenemase and to belong to clade 1 of sequence type 258. Genotyping of the conserved wzi and wzc genes from strain KK207-2 yielded contrasting results: the wzc-based method assigned the cps207-2 to a new K-type, while the wzi-based method assigned it to the known K41 K-type. In order to resolve this contradiction, the capsular polysaccharide of K. pneumoniae KK207-2 was purified and its structure determined by using GLC-MS of appropriate carbohydrate derivatives, ESI-MS of both partial hydrolysis and Smith degradation derived oligosaccharides, and NMR spectroscopy of oligosaccharides, and the lithium degraded, native and de-O-acetylated polysaccharide. All the collected data demonstrated the following repeating unit for the K. pneumoniae KK207-2 capsular polysaccharide: The polysaccharide contains about 0.60 acetyl groups per repeating unit on C6 of the Gal residue. The reactions catalyzed by each glycosyltransferase in the cpsKK207-2 gene cluster were assigned on the basis of structural homology with other Klebsiella K antigens.


Assuntos
Cápsulas Bacterianas/química , Glicosiltransferases/química , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Polissacarídeos Bacterianos/química , Glicosiltransferases/metabolismo , Hidrólise , Espectroscopia de Ressonância Magnética , Polissacarídeos Bacterianos/isolamento & purificação , Polissacarídeos Bacterianos/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Relação Estrutura-Atividade
18.
Nat Chem ; 11(3): 229-236, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30792508

RESUMO

An automated platform that can synthesize a wide range of complex carbohydrates will greatly increase their accessibility and should facilitate progress in glycoscience. Here we report a fully automated process for enzyme-mediated oligosaccharide synthesis that can give easy access to different classes of complex glycans including poly-N-acetyllactosamine derivatives, human milk oligosaccharides, gangliosides and N-glycans. Our automated platform uses a catch and release approach in which glycosyltransferase-catalysed reactions are performed in solution and product purification is accomplished by solid phase extraction. We developed a sulfonate tag that can easily be installed and enables highly efficient solid phase extraction and product release using a single set of washing conditions, regardless of the complexity of the glycan. Using this custom-built synthesizer, as many as 15 reaction cycles can be performed in an automated fashion without a need for lyophilization or buffer exchange steps.


Assuntos
Técnicas de Química Sintética/métodos , Glicosiltransferases/metabolismo , Oligossacarídeos , Sequência de Carboidratos , Gangliosídeos , Humanos , Leite Humano , Oligossacarídeos/síntese química , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Polissacarídeos , Extração em Fase Sólida
19.
Mol Biol Rep ; 46(2): 2153-2175, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30734172

RESUMO

Stone cells are a characteristic trait of pear fruits, and excessive stone cell formation has a significant negative impact on the texture and flavour of the pulp. Lignin is one of the main components of stone cells. Family-1 uridine diphosphate-glycosyltransferases (UGTs) are responsible for the glycosylation modification of monolignols. However, information remains limited regarding the relationship between UGTs and stone cell formation. To address this problem, we identified 139 UGTs from the pear genome, which were distributed in 15 phylogenetic groups (A-M, O, and P). We also performed a collinearity analysis of UGTs among four Rosaceae plants (pear, peach, mei, and strawberry). Phylogenetic analysis suggested that 13 PbUGTs might be related to the glycosylation of monolignols. Analysis of expression patterns demonstrated that most putative monolignol glycosylation-related PbUGTs not only showed high expression levels in flowers and buds but were also induced by exogenous ABA, SA, and MeJA. In addition, the transcript level of Pbr005014.1 (named PbUGT72AJ2) was consistent with the changing trend of lignin content in pear fruit, and the transcript level was also higher in 'Dangshan Su' pear with higher lignin and stone cell contents. Subcellular localization results showed that PbUGT72AJ2 was located mainly in the cytomembrane and cytoplasm. Based on our study, PbUGT72AJ2 is considered to be a monolignol glycosylation-related UGT. Our results provide an important source for the identification of UGTs and a foundation for the future understanding and manipulation of lignin metabolism and stone cell formation in pear fruit.


Assuntos
Glicosiltransferases/genética , Pyrus/genética , Sementes/genética , Frutas/genética , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica de Plantas/genética , Glicosiltransferases/metabolismo , Lignina/genética , Lignina/metabolismo , Família Multigênica , Filogenia , Proteínas de Plantas/genética , Pyrus/metabolismo , Transcriptoma/genética
20.
Glycobiology ; 29(4): 285-287, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30759212

RESUMO

In 2017, we reported a new database on glycosyltransferase (GT) activities, CSDB_GT (http://csdb.glycoscience.ru/gt.html), which was built at the platform of the Carbohydrate Structure Database (CSDB, http://csdb.glycoscience.ru/database/index.html) and contained data on experimentally confirmed GT activities from Arabidopsis thaliana. All entries in CSDB_GT are curated manually upon the analysis of scientific publications, and the key features of the database are accurate structural, genetic, protein and bibliographic references and close-to-complete coverage on experimentally proven GT activities in selected species. In 2018, CSDB_GT was supplemented with data on Escherichia coli GT activities. Now it contains ca. 800 entries on E. coli GTs, including ca. 550 entries with functions predicted in silico. This information was extracted from research papers published up to the year 2018 or was obtained by the authors' efforts on GT annotation. Thus, CSDB_GT was extended to provide not only experimentally confirmed GT activities, but also those predicted on the basis of gene or protein sequence homology that could carry valuable information. Accordingly, a new confirmation status-predicted in silico-was introduced. In addition, the coverage on A. thaliana was extended up to ca. 900 entries, all of which had experimental confirmation. Currently, CSDB_GT provides close-to-complete coverage on experimentally confirmed GT activities from A. thaliana and E. coli presented up to the year 2018.


Assuntos
Carboidratos/química , Bases de Dados de Proteínas , Escherichia coli/enzimologia , Glicosiltransferases/química , Configuração de Carboidratos , Glicosiltransferases/metabolismo
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