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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.
Carbohydr Polym ; 217: 232-239, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31079681

RESUMO

Heparan sulfate (HS) and heparin, representative members of the glycosaminoglycans, possess distinct biological functions in terms of their specific interactions with hundreds of binding proteins. However, the structural properties of HS and heparin are complex due to their variable repeating motifs, different chain lengths and sulfation patterns. A concise chemoenzymatic approach has been developed to obtain well-defined low molecular weight (LMW) HS analogues. Pasteurella multocida heparosan synthase-2 (PmHS2) was utilized to fabricate the HS backbones with controllable chain lengths ranging from 14mer to 26mer. Moreover, regioselective and overall sulfation were conducted by chemical approach. The persulfated HS analogues exhibited more potent beta-site amyloid precursor protein (APP)-cleaving enzyme-1 (BACE-1) inhibitory activity than heparin and enoxaparin, and enhanced BACE-1 inhibitions were also found with the increasing molecular size of the HS analogues. This approach supplies the promising LMW HS analogues for the potential development of novel anti-Alzheimer's drugs.


Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Heparitina Sulfato/análogos & derivados , Inibidores de Proteases/química , Sequência de Carboidratos , Glicosiltransferases/química , Heparitina Sulfato/síntese química , Humanos , Peso Molecular , Pasteurella multocida/enzimologia , Inibidores de Proteases/síntese química
3.
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
4.
Methods Mol Biol ; 1954: 137-150, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30864129

RESUMO

Glycosyltransferases in bacteria are built using only four known architectures, but this structural core is often supplemented by fusions with a wide variety of other domains, including those that help recruit them to the membrane. Structural and functional characterization of these proteins is often simplified by making a subconstruct that is better behaved in solution, and perhaps monofunctional. In this chapter we review bioinformatics tools and strategies that can be used for designing such constructs of glycosyltransferases.


Assuntos
Bactérias/enzimologia , Glicosiltransferases/química , Proteômica/métodos , Software , Bactérias/química , Cristalização/métodos , Internet , Proteínas Intrinsicamente Desordenadas/química , Conformação Proteica
5.
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
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.
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
8.
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
9.
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
10.
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
11.
Int J Biol Macromol ; 128: 101-106, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30664967

RESUMO

A new capsular polysaccharide (CPS) biosynthesis gene cluster, KL16, was found in the genome sequence of a clinical Acinetobacter baumannii ST25 isolate, D4. The variable part of KL16 contains a module of genes for synthesis of 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-non-2-ulosonic acid (5,7-di-N-acetylpseudaminic acid, Pse5Ac7Ac), a gene encoding ItrA3 that initiates the CPS synthesis with d-GlcpNAc, and two glycosyltransferase (Gtr) genes. The K16 CPS was studied by sugar analysis and Smith degradation along with 1D and 2D 1H and 13C NMR spectroscopy, and shown to be built up of linear trisaccharide repeats containing d-galactose (d-Gal), N-acetyl-d-glucosamine (d-GlcNAc), and Pse5Ac7Ac. The d-Galp residue is linked to the d-GlcpNAc initiating sugar via a ß-(1 → 3) linkage evidently formed by a Gtr5 variant, Gtr5K16, encoded in KL16. This reveals an altered or relaxed substrate specificity of this variant as the majority of Gtr5-type glycosyltransferases have previously been shown to form a ß-d-Galp-(1 → 3)-d-GalpNAc linkage. The ß-Psep5Ac7Ac-(2 → 4)-d-Galp linkage is predicted to be formed by the other glycosyltransferase, Gtr37, which does not match members of any known glycosyltransferase family.


Assuntos
Acinetobacter baumannii/genética , Acinetobacter baumannii/metabolismo , Cápsulas Bacterianas/metabolismo , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Família Multigênica , Polissacarídeos Bacterianos/biossíntese , Regulação Bacteriana da Expressão Gênica , Ordem dos Genes , Genes Bacterianos , Ligação Genética , Loci Gênicos , Glicosiltransferases/química , Espectroscopia de Ressonância Magnética , Polissacarídeos Bacterianos/química
12.
Carbohydr Res ; 473: 82-87, 2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30648623

RESUMO

N-glycosyltransferase (NGT) is responsible for transferring hexose monosaccharides to the asparagine side chain of proteins and polypeptides in the consensus sequon (N-(X≠P)-T/S) with nucleotide-activated sugars as donor substrates. Here, we expressed and purified four different N-glycosyltransferases derived from diverse bacteria, including Actinobacillus pleuropneumoniae, Aggregatibacter aphrophilus, Kingella kingae and Bibersteinia trehalosi, and measured their catalytic activities of four synthesized peptides via in vitro glycosylation assays. RP-HPLC and mass spectrometry were used to identify and quantify the glycopeptide formation by distinct NGT isoforms. We then analyzed and compared the glycosylation efficiencies of different peptides for these four NGT isoforms, which showed distinct substrate selectivities. We sought to probe peptide specificities among various NGT isoforms, which could broaden the application of NGT-catalyzed N-glycosylation of a variety of therapeutic proteins.


Assuntos
Glicosiltransferases/metabolismo , Peptídeos/metabolismo , Sequência de Aminoácidos , Bactérias/enzimologia , Glicosiltransferases/química , Isoenzimas/química , Isoenzimas/metabolismo , Especificidade por Substrato , Açúcares/metabolismo
13.
J Microbiol Biotechnol ; 29(2): 268-273, 2019 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-30602272

RESUMO

The specificity of a Bacillus licheniformis uridine diphosphate (UDP) glycosyltransferase, YjiC, was increased towards thymidine diphosphate (TDP)-sugar by site-directed mutagenesis. The Arg-282 of YjiC was identified and investigated by substituting with Trp. Conversion rate and kinetic parameters were compared between YjiC and its variants with several acceptor substrates such as 7-hydroxyflavone (7-HF), 4',7-dihydroxyisoflavone, 7,8-dihydroxyflavone and curcumin. Molecular docking of TDP-glucose and 7-HF with YjiC model showed pi-alkyl interaction with Arg-282 and His-14, and pi-pi interaction with His14 and thymine ring. YjiC (H14A) variant lost its glucosylation activity with TDP-glucose validating significance of His-14 in binding of TDP-sugars.


Assuntos
Bacillus licheniformis/enzimologia , Glicosiltransferases/química , Glicosiltransferases/metabolismo , Açúcares de Nucleosídeo Difosfato/metabolismo , Uridina Difosfato Glucose/metabolismo , Sequência de Aminoácidos , Bacillus licheniformis/genética , Sítios de Ligação/genética , Flavonoides/metabolismo , Glicosilação , Glicosiltransferases/genética , Cinética , Simulação de Acoplamento Molecular , Mutação , Engenharia de Proteínas , Relação Estrutura-Atividade , Especificidade por Substrato/genética
14.
Phytochemistry ; 157: 135-144, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30399496

RESUMO

The UDP-glycosyltransferase UGT88F subfamily has been described first in Malus x domestica with the characterization of UGT88F1. Up to now UGT88F1 was one of the most active UGT glycosylating dihydrochalcones in vitro. The involvement of UGT88F1 in phloridzin (phloretin 2'-O-glucoside) synthesis, the main apple tree dihydrochalcone, was further confirmed in planta. Since the characterization of UGT88F1, this new UGT subfamily has been poorly studied probably because it seemed restricted to Maloideae. In the present study, we investigate the apple tree genome to identify and biochemically characterize the whole UGT88F subfamily. The apple tree genome contains five full-length UGT88F genes out of which three newly identified members (UGT88F6, UGT88F7 and UGT88F8) and a pseudogene. These genes are organized into two genomic clusters resulting from the recent global genomic duplication event in the apple tree. We show that recombinant UGT88F8 protein specifically glycosylates phloretin in the 2'OH position to synthetize phloridzin in vitro and was therefore named UDP-glucose: phloretin 2'-O-glycosyltransferase. The Km values of UGT88F8 are 7.72 µM and 10.84 µM for phloretin and UDP-glucose respectively and are in the same range as UGT88F1 catalytic parameters thus constituting two isoforms. Co-expression patterns of both UGT88F1 and UGT88F8 argue for a redundant function in phloridzin biosynthesis in planta. Contrastingly, recombinant UGT88F6 protein is able to glycosylate in vitro a wide range of flavonoids including flavonols, flavones, flavanones, chalcones and dihydrochalcones, although flavonols are the preferred substrates, e.g. Km value for kaempferol is 2.1 µM. Depending on the flavonoid, glycosylation occurs at least on the 3-OH and 7-OH positions. Therefore UGT88F6 corresponds to an UDP-glucose: flavonoid 3/7-O-glycosyltransferase. Finally, a molecular modeling study highlights a very high substitution rate of residues in the acceptor binding pocket between UGT88F8 and UGT88F6 which is responsible for the enzymes divergence in substrate and regiospecificity, despite an overall high protein homology.


Assuntos
Genômica , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Malus/enzimologia , Malus/genética , Genoma de Planta/genética , Glicosiltransferases/química , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Conformação Proteica , Temperatura Ambiente
15.
Phytochemistry ; 157: 184-193, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30419412

RESUMO

UDP-dependent glycosyltransferases (UGTs) convert aglycones into more stable, bioactive, and structurally diverse glycosylated derivatives. Pomegranate (Punica granatum L.) produces various glycosylated phenolic metabolites, e.g. hydrolyzable tannins (HTs), anthocyanins, and flavonoids, and constitutes an excellent system for investigating the corresponding UGT activities. Here we report the cloning and functional characterization of a pomegranate UGT, PgUGT95B2, which is highly active towards flavones and flavonols and can glycosylate at more than one position in the substrate molecule. Particularly, PgUGT95B2 has the strongest activity towards tricetin (flavone with a tri-hydroxylated B-ring) and can act at the 4'-O position of its B-ring. In addition, PgUGT95B2 was able to glycosylate flavones present in pomegranate metabolite extracts. Conversely, PgUGT95B2 did not produce a galloylglucose ester (precursor for HT biosynthesis) or anthocyanins in enzyme assays. Our phylogenetic analysis suggested an independent evolution of PgUGT95B2 and flavone/flavonol UGTs identified in the model plant Arabidopsis thaliana through convergent evolution or gene loss.


Assuntos
Arabidopsis/enzimologia , Evolução Molecular , Flavonas/metabolismo , Flavonóis/metabolismo , Glicosiltransferases/metabolismo , Punicaceae/enzimologia , Glicosiltransferases/química , Ligações de Hidrogênio , Hidrólise , Simulação de Acoplamento Molecular , Filogenia , Conformação Proteica , Especificidade por Substrato
16.
J Agric Food Chem ; 66(44): 11701-11708, 2018 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-30350978

RESUMO

Crocins are the most important active ingredient found in Crocus sativus, a well-known "plant gold". The glycosyltransferase-catalyzed glycosylation of crocetin is the last step of biosynthesizing crocins and contributes to their structural diversity. Crocin biosynthesis is now hampered by the lack of efficient glycosyltransferases with activity toward crocetin. In this study, two microbial glycosyltransferases (Bs-GT and Bc-GTA) were successfully mined based on the comprehensive analysis of the PSPG motif and the N-terminal motif of the target plant-derived UGT75L6 and Cs-GT2. Bs-GT from Bacillus subtilis 168, an enzyme with a higher activity of glycosylation toward crocetin than that of Bc-GTA, was characterized. The efficient synthesis of crocins from crocetin catalyzed by microbial GT (Bs-GT) was first reported with a high molecular conversion rate of 81.9%, resulting in the production of 476.8 mg/L of crocins. The glycosylation of crocetin on its carboxyl groups by Bs-GT specifically produced crocin-5 and crocin-3, the important rare crocins.


Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , Carotenoides/química , Glicosiltransferases/química , Glicosilação
17.
World J Microbiol Biotechnol ; 34(10): 150, 2018 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-30255239

RESUMO

This work used an approach of enzyme engineering towards the improved production of baicalin as well as alteration of acceptor and donor substrate preferences in UGT73A16. The 3D model of Withania somnifera family-1 glycosyltransferase (UGT73A16) was constructed based on the known crystal structures of plant UGTs. Structural and functional properties of UGT73A16 were investigated using docking and mutagenesis. The docking studies were performed to understand the key residues involved in substrate recognition. In the molecular model of UGT73A16, substrates binding pockets are located between N- and C-terminal domains. Modeled UGT73A16 was docked with UDP-glucose, UDP-glucuronic acid (UDPGA), kaempferol, isorhamnetin, 3-hydroxy flavones, naringenin, genistein and baicalein. The protein-ligand interactions showed that His 16, Asp 246, Lys 255, Ala 337, Gln 339, Val 340, Asn 358 and Glu 362 amino acid residues may be important for catalytic activity. The kinetic parameters indicated that mutants A337C and Q339A exhibited 2-3 fold and 6-7 fold more catalytic efficiency, respectively than wild type, and shifted the sugar donor specificity from UDP-glucose to UDPGA. The mutant Q379H displayed large loss of activity with UDP-glucose and UDPGA strongly suggested that last amino acid residue of PSPG box is important for glucuronosylation and glucosylation and highly specific to sugar binding sites. The information obtained from docking and mutational studies could be beneficial in future to engineer this biocatalyst for development of better ones.


Assuntos
Glicosiltransferases/química , Glicosiltransferases/metabolismo , Mutagênese , Withania/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Flavonoides , Cinética , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Conformação Proteica , Alinhamento de Sequência , Análise de Sequência , Homologia de Sequência de Aminoácidos
18.
Int J Mol Sci ; 19(8)2018 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-30104475

RESUMO

Next-generation sequencing (NGS) of the Flammulina elastica (wood-rotting basidiomycete) genome was performed to identify carbohydrate-active enzymes (CAZymes). The resulting assembly (31 kmer) revealed a total length of 35,045,521 bp (49.7% GC content). Using the AUGUSTUS tool, 12,536 total gene structures were predicted by ab initio gene prediction. An analysis of orthologs revealed that 6806 groups contained at least one F. elastica protein. Among the 12,536 predicted genes, F. elastica contained 24 species-specific genes, of which 17 genes were paralogous. CAZymes are divided into five classes: glycoside hydrolases (GHs), carbohydrate esterases (CEs), polysaccharide lyases (PLs), glycosyltransferases (GTs), and auxiliary activities (AA). In the present study, annotation of the predicted amino acid sequences from F. elastica genes using the dbCAN CAZyme database revealed 508 CAZymes, including 82 AAs, 218 GHs, 89 GTs, 18 PLs, 59 CEs, and 42 carbohydrate binding modules in the F. elastica genome. Although the CAZyme repertoire of F. elastica was similar to those of other fungal species, the total number of GTs in F. elastica was larger than those of other basidiomycetes. This genome information elucidates newly identified wood-degrading machinery in F. elastica, offers opportunities to better understand this fungus, and presents possibilities for more detailed studies on lignocellulosic biomass degradation that may lead to future biotechnological and industrial applications.


Assuntos
Flammulina/genética , Proteínas Fúngicas/genética , Genoma Fúngico , Bases de Dados Genéticas , Flammulina/enzimologia , Proteínas Fúngicas/classificação , Proteínas Fúngicas/metabolismo , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Glicosiltransferases/química , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Filogenia , Polissacarídeo-Liase/química , Polissacarídeo-Liase/genética , Polissacarídeo-Liase/metabolismo , Análise de Sequência de DNA , Sequenciamento Completo do Genoma
19.
J Agric Food Chem ; 66(36): 9446-9455, 2018 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-30095259

RESUMO

Ginsenosides attract great attention for their bioactivities. However, their contents are low, and many UDP-glycosyltransferases (UGTs) that play crucial roles in the ginsenoside biosynthesis pathways have not been identified, which hinders the biosynthesis of ginsenosides. In this study, we reported that one UDP-glycosyltransferase, UGTPg71A29, from Panax ginseng could glycosylate C20-OH of Rh1 and transfer a glucose moiety to Rd, producing ginsenosides Rg1 and Rb1, respectively. Ectopic expression of UGTPg71A29 in Saccharomyces cerevisiae stably generated Rg1 and Rb1 under its corresponding substrate. Overexpression of UGTPg71A29 in transgenic cells of P. ginseng could significantly enhance the accumulation of Rg1 and Rb1, with their contents of 3.2- and 3.5-fold higher than those in the control, respectively. Homology modeling, molecular dynamics, and mutational analysis revealed the key catalytic site, Gln283, which provided insights into the catalytic mechanism of UGTPg71A29. These results not only provide an efficient enzymatic tool for the synthesis of glycosides but also help achieve large-scale industrial production of glycosides.


Assuntos
Ginsenosídeos/biossíntese , Glicosiltransferases/química , Glicosiltransferases/metabolismo , Panax/enzimologia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Motivos de Aminoácidos , Vias Biossintéticas , Catálise , Domínio Catalítico , Glicosiltransferases/genética , Simulação de Dinâmica Molecular , Panax/química , Panax/genética , Proteínas de Plantas/genética , Difosfato de Uridina/metabolismo
20.
Molecules ; 23(7)2018 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-30004451

RESUMO

Glycosylamines are valuable sugar derivatives that have attracted much attention as synthetic intermediates en route to iminosugar-C-glycosyl compounds. Iminosugars are among the most important glycomimetics reported to date due to their powerful activities as inhibitors of a wide variety of glycosidases and glycosyltransferases, as well as for their use as pharmacological chaperones. As they provide ready access to these important glycoside mimics, we have reviewed the most significant glycosylamine-based methodologies developed to date, with a special emphasis on the literature reported after 2006. The groups of substrates covered include N-alkyl- and N-benzyl-glycosylamines, N-glycosylhydroxylamines, N-(alkoxycarbonyl)-, and N-tert-butanesulfinyl-glycosylamines.


Assuntos
Glicosídeos/química , Inibidores Enzimáticos/química , Glicosídeo Hidrolases/química , Glicosiltransferases/química
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