Analysis of the diversity of the glycoside hydrolase family 130 in mammal gut microbiomes reveals a novel mannoside-phosphorylase function
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PROVIDER: PRJEB49017 | ENA |
REPOSITORIES: ENA
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Project description:Mannoside phosphorylases are involved in the intracellular metabolization of mannooligosaccharides, and are also useful enzymes for the in vitro synthesis of oligosaccharides. They are found in glycoside hydrolase family GH130. Here we report on an analysis of 6308 GH130 sequences, including 4714 from the human, bovine, porcine and murine microbiomes. Using sequence similarity networks, we divided the diversity of sequences into 15 mostly isofunctional meta-nodes; of these, 9 contained no experimentally characterized member. By examining the multiple sequence alignments in each meta-node, we predicted the determinants of the phosphorolytic mechanism and linkage specificity. We thus hypothesized that eight uncharacterized meta-nodes would be phosphorylases. These sequences are characterized by the absence of signal peptides and of the catalytic base. Those sequences with the conserved E/K, E/R and Y/R pairs of residues involved in substrate binding would target β-1,2-, β-1,3- and β-1,4-linked mannosyl residues, respectively. These predictions were tested by characterizing members of three of the uncharacterized meta-nodes from gut bacteria. We discovered the first known β-1,4-mannosyl-glucuronic acid phosphorylase, which targets a motif of the Shigella lipopolysaccharide O-antigen. This work uncovers a reliable strategy for the discovery of novel mannoside-phosphorylases, reveals possible interactions between gut bacteria, and identifies a biotechnological tool for the synthesis of antigenic oligosaccharides.
| S-EPMC7660257 | biostudies-literature
Project description:Proteomic data from supernatants from thermophilic cellulolytic microbial communities, for the identification of specific hydrolases from Thermobispora bispora related actinobacterium.
2016-08-25 | PXD004204 | Pride
Project description:Sucrose phosphorylases are carbohydrate-active enzymes with outstanding potential for the biocatalytic conversion of common table sugar into products with attractive properties. They belong to the glycoside hydrolase family GH13, where they are found in subfamily 18. In bacteria, these enzymes catalyse the phosphorolysis of sucrose to yield α-glucose 1-phosphate and fructose. However, sucrose phosphorylases can also be applied as versatile transglucosylases for the synthesis of valuable glycosides and sugars because their broad promiscuity allows them to transfer the glucosyl group of sucrose to a diverse collection of compounds other than phosphate. Numerous process and enzyme engineering studies have expanded the range of possible applications of sucrose phosphorylases ever further. Moreover, it has recently been discovered that family GH13 also contains a few novel phosphorylases that are specialised in the phosphorolysis of sucrose 6F-phosphate, glucosylglycerol or glucosylglycerate. In this review, we provide an overview of the progress that has been made in our understanding and exploitation of sucrose phosphorylases and related enzymes over the past ten years.
| S-EPMC7178133 | biostudies-literature
Project description:Few aerobic hyperthermophiles degrade polysaccharides. We describe the genome-enabled enrichment and isolation of an aerobic hyperthermophile, Fervidibacter sacchari, which was originally ascribed to candidate phylum Fervidibacteria. F. sacchari uses polysaccharides and monosaccharides as sole carbon sources from 65-87.5 °C, and its genome encodes 117 glycoside hydrolases (GHs) spanning 49 GH families, including 31 homologs of understudied GH109, GH177, and GH179 domains. Here, we analyzed the transcriptomes of F. sacchari cells grown on eight different sole carbon and energy sources (beta-glucan, chondroitin sulfate, corn stover, gellan gum, locust bean gum, starch, xanthan gum, and xyloglucan) to link glycoside hydrolase substrate to function, as well as identify potential regulatory mechanisms. These data will provide preliminary characterization of novel carbohydrate-active enzymes at high temperatures.
2023-12-31 | GSE249938 | GEO
Project description:Functional exploration of novel glycoside hydrolases in Fervidibacter sacchari PD1T
| PRJNA1051158 | ENA
Project description:Glycoside phosphorylases (EC 2.4.x.x) carry out the reversible phosphorolysis of glucan polymers, producing the corresponding sugar 1-phosphate and a shortened glycan chain. ?-1,3-Glucan phosphorylase activities have been reported in the photosynthetic euglenozoan Euglena gracilis, but the cognate protein sequences have not been identified to date. Continuing our efforts to understand the glycobiology of E. gracilis, we identified a candidate phosphorylase sequence, designated EgP1, by proteomic analysis of an enriched cellular protein lysate. We expressed recombinant EgP1 in Escherichia coli and characterized it in vitro as a ?-1,3-glucan phosphorylase. BLASTP identified several hundred EgP1 orthologs, most of which were from Gram-negative bacteria and had 37-91% sequence identity to EgP1. We heterologously expressed a bacterial metagenomic sequence, Pro_7066 in E. coli and confirmed it as a ?-1,3-glucan phosphorylase, albeit with kinetics parameters distinct from those of EgP1. EgP1, Pro_7066, and their orthologs are classified as a new glycoside hydrolase (GH) family, designated GH149. Comparisons between GH94, EgP1, and Pro_7066 sequences revealed conservation of key amino acids required for the phosphorylase activity, suggesting a phosphorylase mechanism that is conserved between GH94 and GH149. We found bacterial GH149 genes in gene clusters containing sugar transporter and several other GH family genes, suggesting that bacterial GH149 proteins have roles in the degradation of complex carbohydrates. The Bacteroidetes GH149 genes located to previously identified polysaccharide utilization loci, implicated in the degradation of complex carbohydrates. In summary, we have identified a eukaryotic and a bacterial ?-1,3-glucan phosphorylase and uncovered a new family of phosphorylases that we name GH149.
| S-EPMC5827456 | biostudies-literature
Project description:Phylogenetic, microbiological and comparative genomic analysis was used to examine the diversity among members of the genus Caldicellulosiruptor with an eye towards the capacity of these extremely thermophilic bacteria for degrading the complex carbohydrate content of plant biomass. Seven species from this genus (C. saccharolyticus, C. bescii (formerly Anaerocellum thermophilum), C. hydrothermalis, C. owensensis, C. kronotskyensis, C. lactoaceticus, and C. kristjanssonii) were compared on the basis of 16S rRNA phylogeny and cross-species DNA-DNA hybridization to a whole genome C. saccharolyticus oligonucleotide microarray. Growth physiology of the seven Caldicellulosiruptor species on a range of carbohydrates showed that, while all could be cultivated on acid pre-treated switchgrass, only C. saccharolyticus, C. besci, C. kronotskyensis, and C. lactoaceticus were capable of hydrolyzing Whatman No. 1 filter paper. Two-dimensional gel electrophoresis of the secretomes from cells grown on microcrystalline cellulose revealed that species capable of crystalline cellulose hydrolysis also had diverse secretome fingerprints. The two-dimensional secretome of C. saccharolyticus revealed a prominent S-layer protein that appears to be also indicative of highly cellulolytic Caldicellulosiruptor species, suggesting a possible role in cell-substrate interaction. These growth physiology results were also linked to glycoside hydrolase and carbohydrate-binding module inventories for the seven bacteria, deduced from draft genome sequence information. These preliminary inventories indicated that the absence of a single glycoside hydrolase family and carbohydrate binding motif family appear to be responsible for some Caldicellulosiruptor species’ diminished cellulolytic capabilities. Overall, the genus Caldicellulosiruptor appears to contain more genomic and physiological diversity than previously reported, and is well suited for biomass deconstruction applications. Six dye-flip experiments were conducted using C. saccharolyticus genomic DNA as the reference in each dye-flip, and one of six different Caldicellulosiruptor spp. as a tester in each dye-flip
2010-11-29 | E-GEOD-23606 | biostudies-arrayexpress
Project description:Phylogenetic, microbiological and comparative genomic analysis was used to examine the diversity among members of the genus Caldicellulosiruptor with an eye towards the capacity of these extremely thermophilic bacteria for degrading the complex carbohydrate content of plant biomass. Seven species from this genus (C. saccharolyticus, C. bescii (formerly Anaerocellum thermophilum), C. hydrothermalis, C. owensensis, C. kronotskyensis, C. lactoaceticus, and C. kristjanssonii) were compared on the basis of 16S rRNA phylogeny and cross-species DNA-DNA hybridization to a whole genome C. saccharolyticus oligonucleotide microarray. Growth physiology of the seven Caldicellulosiruptor species on a range of carbohydrates showed that, while all could be cultivated on acid pre-treated switchgrass, only C. saccharolyticus, C. besci, C. kronotskyensis, and C. lactoaceticus were capable of hydrolyzing Whatman No. 1 filter paper. Two-dimensional gel electrophoresis of the secretomes from cells grown on microcrystalline cellulose revealed that species capable of crystalline cellulose hydrolysis also had diverse secretome fingerprints. The two-dimensional secretome of C. saccharolyticus revealed a prominent S-layer protein that appears to be also indicative of highly cellulolytic Caldicellulosiruptor species, suggesting a possible role in cell-substrate interaction. These growth physiology results were also linked to glycoside hydrolase and carbohydrate-binding module inventories for the seven bacteria, deduced from draft genome sequence information. These preliminary inventories indicated that the absence of a single glycoside hydrolase family and carbohydrate binding motif family appear to be responsible for some Caldicellulosiruptor species’ diminished cellulolytic capabilities. Overall, the genus Caldicellulosiruptor appears to contain more genomic and physiological diversity than previously reported, and is well suited for biomass deconstruction applications.
2010-11-29 | GSE23606 | GEO
Project description:Purpose: Guided by an in silico combination of microRNA (miRNA) target prediction, analysis of transcriptomic changes in 137 human diseases, and advanced gene network modeling, we predicted the miR-130/301 family of miRNAs as a shared regulator of a fibrotic gene network across human diseases, thus orchestrating broad control over disease manifestation. The goals of this study are to compare the lung mRNA profile of mouse model of Pulmonary hypertension, one of the most fibrotic pathology uncovered by our in silico prediction, treated with an inhibitor of miR-130/301 (Short-130) to mice treated with a control inhibitor (Short-NC). Methods: Eight-week-old mice (C57BL/6) were injected with SU5416 (20 mg/kg/dose; Sigma-Aldrich), followed by exposure to normobaric hypoxia (10% O2; OxyCycler chamber, Biospherix Ltd.) for 2 weeks. After 2 weeks and confirmation of PH development in 5 mice (right heart catheterization), mice were further treated with 3 intrapharyngeal injections (every 4 days) of control or miR-130/301 shortmer oligonucleotides, designed as fully modified antisense oligonucleotides complementary to the seed sequence of the miR-130/301 miRNA family (10 mg/kg/dose; Regulus). Specifically, the control and miR-130/301 shortmer oligonucleotides were nontoxic, lipid-permeable, high-affinity oligonucleotides. The miR-130/301 shortmer carried a sequence complementary to the active site of the miR-130/301 miRNA family, containing a phosphorothioate backbone and modifications (fluoro-, methoxyethyl, and bicyclic sugar) at the sugar 2’ position. Three days after the last injection, right heart catheterization was performed followed by harvesting of lung tissue for RNA extraction. Lung mRNA profiles of those mice or control mice (Normoxia+SU5416) were generated by deep sequencing, in triplicate, using Illumina HiSeq 2000. The sequence reads that passed quality filters were analyzed at the gene-level count. The gene level counts were then normalized with the R/Bioconductor package limma using the voom /variance stabilization method. The data were quality controlled for outliers using principal component analysis (PCA). Differential expression analysis between transcriptome profiles of experimental groups was performed using the R / Bioconductor package limma. Results: Transcriptomic analyses of whole lung from mice with hypoxia+SU5416-induced PH revealed a generalized de-repression of miR-130/301 targets by Short-130 treatment. Importantly, although whole lung transcriptomics likely captured only a subset of the miR-130/301 targets affecting the diseased pulmonary vasculature, pathway enrichment nonetheless revealed pronounced representation of several pathways known to be involved in fibrosis. Thus, the miR-130/301 family indeed induces a programmatic shift at the molecular level toward the fibrotic pathophenotype in vivo
2015-09-29 | GSE61828 | GEO
Project description:We synthesized and evaluated new zwitterionic inhibitors against glycoside hydrolase-like phosphorylase Streptomyces coelicolor (Sco) GlgEI-V279S which plays a role in ?-glucan biosynthesis. Sco GlgEI-V279S serves as a model enzyme for validated anti-tuberculosis (TB) target Mycobacterium tuberculosis (Mtb) GlgE. Pyrrolidine inhibitors 5 and 6 were designed based on transition state considerations and incorporate a phosphonate on the pyrrolidine moiety to expand the interaction network between the inhibitor and the enzyme active site. Compounds 5 and 6 inhibited Sco GlgEI-V279S with Ki = 45 ± 4 ?M and 95 ± 16 ?M, respectively, and crystal structures of Sco GlgE-V279S-5 and Sco GlgE-V279S-6 were obtained at a 3.2 Å and 2.5 Å resolution, respectively.
| S-EPMC6169318 | biostudies-literature