In situ visualization of glycoside hydrolase family 92 genes in marine flavobacteria

AbstractGene clusters rich in carbohydrate-active enzymes within Flavobacteriia genera provide a competitiveness for their hosts to degrade diatom-derived polysaccharides. One such widely distributed polysaccharide is glucuronomannan, a main cell wall component of diatoms. A conserved gene cluster putatively degrading glucuronomannan was found previously among various flavobacterial taxa in marine metagenomes. Here, we aimed to visualize two glycoside hydrolase family 92 genes coding for α-mannosidases with fluorescently-labeled polynucleotide probes using direct-geneFISH. Reliable in situ localization of single-copy genes was achieved with an efficiency up to 74% not only in the flavobacterial strains Polaribacter Hel1_33_49 and Formosa Hel1_33_131 but also in planktonic samples from the North Sea. In combination with high-resolution microscopy, direct-geneFISH gave visual evidence of the contrasting lifestyles of closely related Polaribacter species in those samples and allowed for the determination of gene distribution among attached and free-living cells. We also detected highly similar GH92 genes in yet unidentified taxa by broadening probe specificities, enabling a visualization of the functional trait in subpopulations across the borders of species and genera. Such a quantitative insight into the niche separation of flavobacterial taxa complements our understanding of the ecology of polysaccharide-degrading bacteria beyond omics-based techniques on a single-cell level.

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A chemo-enzymatic approach for the synthesis of human milk oligosaccharide backbone structures

Zeitschrift für Naturforschung C ◽

10.1515/znc-2018-0149 ◽

2019 ◽

Vol 74 (3-4) ◽

pp. 85-89 ◽

Cited By ~ 8

Author(s):

Jan Muschiol ◽

Anne S. Meyer

Keyword(s):

Human Milk ◽

Glycoside Hydrolase ◽

Cascade Reaction ◽

Glycoside Hydrolase Family ◽

Donor Molecule ◽

Milk Oligosaccharide ◽

Human Milk Oligosaccharide ◽

First Time ◽

Hydrolase Family

Abstract The ability of an engineered β-N-acetylhexosaminidase to utilize a reactive oxazoline as donor molecule for transglycosylation reaction to synthesize human milk oligosaccharide backbone structures was studied. The human milk oligosaccharide precursor lacto-N-triose II and three regioisomers could be synthesized using the oxazoline, which was either in situ-generated resulting in a chemo-enzymatic sequential cascade or was used as a purified compound. The highest observed concentration of overall transglycosylation products in a cascade reaction was 13.7 mM after 18.5 h, whereas the use of purified oxazoline resulted in 25.0 mM of transglycosylation products after 6.5 h. Remarkably, the in situ-generated oxazoline could be used without any further purification and it was shown that the used enzyme tolerated significant amounts of reagents such as triethylamine, which is reported for the first time for an enzyme from the glycoside hydrolase family 20.

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Characterization and Functional Importance of Two Glycoside Hydrolase Family 16 Genes from the Rice White Tip Nematode Aphelenchoides besseyi

Animals ◽

10.3390/ani11020374 ◽

2021 ◽

Vol 11 (2) ◽

pp. 374

Author(s):

Hui Feng ◽

Dongmei Zhou ◽

Paul Daly ◽

Xiaoyu Wang ◽

Lihui Wei

Keyword(s):

Glycoside Hydrolase ◽

Amino Acid Sequences ◽

Parasitic Nematodes ◽

Glycoside Hydrolase Family ◽

Plant Parasitic Nematodes ◽

Aphelenchoides Besseyi ◽

Rice White Tip Nematode ◽

Hydrolase Family ◽

Plant Parasitic

The glycoside hydrolase family 16 (GH16) is widely found in prokaryotes and eukaryotes, and hydrolyzes the β-1,3(4)-linkages in polysaccharides. Notably, the rice white tip nematode Aphelenchoides besseyi harbors a higher number of GH16s compared with other plant-parasitic nematodes. In this work, two GH16 genes, namely AbGH16-1 and AbGH16-2, were isolated and characterized from A. besseyi. The deduced amino acid sequences of AbGH16-1 and AbGH16-2 contained an N-terminal signal peptide and a fungal Lam16A glucanase domain. Phylogenetic analysis revealed that AbGH16-1 and AbGH16-2 clustered with ascomycete GH16s, suggesting AbGH16-1 and AbGH16-2 were acquired by horizontal gene transfer from fungi. In situ hybridization showed that both AbGH16-1 and AbGH16-2 were specifically expressed in the nematode gonads, correlating with qPCR analysis that showed the high transcript levels of the two genes in the female nematodes. AbGH16-1 and AbGH16-2 were also significantly induced in nematodes feeding on Botrytis cinerea. Characterization of the recombinant protein showed AbGH16-1 and AbGH16-2 displayed pronounced inhibition of both conidial germination and germ tube elongation of B. cinerea. In addition, silencing of AbGH16-1 and AbGH16-2 by RNA interference significantly decreased the reproduction ability of A. besseyi and had a profound impact on the development process of offspring in this nematode. These findings have firstly proved that GH16s may play important roles in A.besseyi feeding and reproduction on fungi, which thus provides novel insights into the function of GH16s in plant-parasitic nematodes.

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Xylanases of glycoside hydrolase family 30 – An overview

Biotechnology Advances ◽

10.1016/j.biotechadv.2021.107704 ◽

2021 ◽

Vol 47 ◽

pp. 107704

Author(s):

Vladimír Puchart ◽

Katarína Šuchová ◽

Peter Biely

Keyword(s):

Glycoside Hydrolase ◽

Glycoside Hydrolase Family ◽

Hydrolase Family ◽

Glycoside Hydrolase Family 30

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Modeled 3D-Structures of Proteobacterial Transglycosylases from Glycoside Hydrolase Family 17 Give Insight in Ligand Interactions Explaining Differences in Transglycosylation Products

Applied Sciences ◽

10.3390/app11094048 ◽

2021 ◽

Vol 11 (9) ◽

pp. 4048

Author(s):

Javier A. Linares-Pastén ◽

Lilja Björk Jonsdottir ◽

Gudmundur O. Hreggvidsson ◽

Olafur H. Fridjonsson ◽

Hildegard Watzlawick ◽

...

Keyword(s):

Glycoside Hydrolase ◽

Ligand Docking ◽

Glycoside Hydrolase Family ◽

Ligand Interactions ◽

Tim Barrel ◽

Branching Point ◽

The Difference ◽

Dynamics Simulations ◽

And Function ◽

Hydrolase Family

The structures of glycoside hydrolase family 17 (GH17) catalytic modules from modular proteins in the ndvB loci in Pseudomonas aeruginosa (Glt1), P. putida (Glt3) and Bradyrhizobium diazoefficiens (previously B. japonicum) (Glt20) were modeled to shed light on reported differences between these homologous transglycosylases concerning substrate size, preferred cleavage site (from reducing end (Glt20: DP2 product) or non-reducing end (Glt1, Glt3: DP4 products)), branching (Glt20) and linkage formed (1,3-linkage in Glt1, Glt3 and 1,6-linkage in Glt20). Hybrid models were built and stability of the resulting TIM-barrel structures was supported by molecular dynamics simulations. Catalytic amino acids were identified by superimposition of GH17 structures, and function was verified by mutagenesis using Glt20 as template (i.e., E120 and E209). Ligand docking revealed six putative subsites (−4, −3, −2, −1, +1 and +2), and the conserved interacting residues suggest substrate binding in the same orientation in all three transglycosylases, despite release of the donor oligosaccharide product from either the reducing (Glt20) or non-reducing end (Glt1, Gl3). Subsites +1 and +2 are most conserved and the difference in release is likely due to changes in loop structures, leading to loss of hydrogen bonds in Glt20. Substrate docking in Glt20 indicate that presence of covalently bound donor in glycone subsites −4 to −1 creates space to accommodate acceptor oligosaccharide in alternative subsites in the catalytic cleft, promoting a branching point and formation of a 1,6-linkage. The minimum donor size of DP5, can be explained assuming preferred binding of DP4 substrates in subsite −4 to −1, preventing catalysis.

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Understanding the Polymerization Mechanism of Glycoside-Hydrolase Family 70 Glucansucrases

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)84038-3 ◽

2006 ◽

Vol 281 (42) ◽

pp. 31254-31267

Author(s):

Claire Moulis ◽

Gilles Joucla ◽

David Harrison ◽

Emeline Fabre ◽

Gabrielle Potocki-Veronese ◽

...

Keyword(s):

Glycoside Hydrolase ◽

Glycoside Hydrolase Family ◽

Polymerization Mechanism ◽

Hydrolase Family

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Endo-fucoidan hydrolases from glycoside hydrolase family 107 (GH107) display structural and mechanistic similarities to α-l-fucosidases from GH29

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.005134 ◽

2018 ◽

Vol 293 (47) ◽

pp. 18296-18308 ◽

Cited By ~ 10

Author(s):

Chelsea Vickers ◽

Feng Liu ◽

Kento Abe ◽

Orly Salama-Alber ◽

Meredith Jenkins ◽

...

Keyword(s):

Glycoside Hydrolase ◽

Biological Activities ◽

Bacterial Species ◽

Structural Data ◽

Side Chain ◽

Glycoside Hydrolase Family ◽

X Ray Crystallography ◽

Catalytic Mechanisms ◽

Thickening Agents ◽

Hydrolase Family

Fucoidans are chemically complex and highly heterogeneous sulfated marine fucans from brown macro algae. Possessing a variety of physicochemical and biological activities, fucoidans are used as gelling and thickening agents in the food industry and have anticoagulant, antiviral, antitumor, antibacterial, and immune activities. Although fucoidan-depolymerizing enzymes have been identified, the molecular basis of their activity on these chemically complex polysaccharides remains largely uninvestigated. In this study, we focused on three glycoside hydrolase family 107 (GH107) enzymes: MfFcnA and two newly identified members, P5AFcnA and P19DFcnA, from a bacterial species of the genus Psychromonas. Using carbohydrate-PAGE, we show that P5AFcnA and P19DFcnA are active on fucoidans that differ from those depolymerized by MfFcnA, revealing differential substrate specificity within the GH107 family. Using a combination of X-ray crystallography and NMR analyses, we further show that GH107 family enzymes share features of their structures and catalytic mechanisms with GH29 α-l-fucosidases. However, we found that GH107 enzymes have the distinction of utilizing a histidine side chain as the proposed acid/base catalyst in its retaining mechanism. Further interpretation of the structural data indicated that the active-site architectures within this family are highly variable, likely reflecting the specificity of GH107 enzymes for different fucoidan substructures. Together, these findings begin to illuminate the molecular details underpinning the biological processing of fucoidans.

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