Glycoside hydrolase family 5: structural snapshots highlighting the involvement of two conserved residues in catalysis

2021 ◽  
Vol 77 (2) ◽  
pp. 205-216
Author(s):  
Laetitia Collet ◽  
Corinne Vander Wauven ◽  
Yamina Oudjama ◽  
Moreno Galleni ◽  
Raphael Dutoit
Keyword(s):  
Catalytic Mechanism ◽  
Glycoside Hydrolases ◽  
Glycoside Hydrolase Family ◽  
Enzymatic Assays ◽  
Conserved Residues ◽  
Displacement Mechanism ◽  
Significant Movement ◽  
Binding Cleft ◽  
Hydrolase Family ◽  
Enzyme Intermediate

The ability of retaining glycoside hydrolases (GHs) to transglycosylate is inherent to the double-displacement mechanism. Studying reaction intermediates, such as the glycosyl-enzyme intermediate (GEI) and the Michaelis complex, could provide valuable information to better understand the molecular factors governing the catalytic mechanism. Here, the GEI structure of RBcel1, an endo-1,4-β-glucanase of the GH5 family endowed with transglycosylase activity, is reported. It is the first structure of a GH5 enzyme covalently bound to a natural oligosaccharide with the two catalytic glutamate residues present. The structure of the variant RBcel1_E135A in complex with cellotriose is also reported, allowing a description of the entire binding cleft of RBcel1. Taken together, the structures deliver different snapshots of the double-displacement mechanism. The structural analysis revealed a significant movement of the nucleophilic glutamate residue during the reaction. Enzymatic assays indicated that, as expected, the acid/base glutamate residue is crucial for the glycosylation step and partly contributes to deglycosylation. Moreover, a conserved tyrosine residue in the −1 subsite, Tyr201, plays a determinant role in both the glycosylation and deglycosylation steps, since the GEI was trapped in the RBcel1_Y201F variant. The approach used to obtain the GEI presented here could easily be transposed to other retaining GHs in clan GH-A.

Structural insights into the substrate specificity and transglycosylation activity of a fungal glycoside hydrolase family 5 β-mannosidase

2014 ◽  
Vol 70 (11) ◽  
pp. 2970-2982 ◽  
Author(s):  
Peng Zhou ◽  
Yang Liu ◽  
Qiaojuan Yan ◽  
Zhongzhou Chen ◽  
Zhen Qin ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Catalytic Mechanism ◽  
Rhizomucor Miehei ◽  
Glycoside Hydrolases ◽  
Glycoside Hydrolase Family ◽  
Transglycosylation Activity ◽  
Potential Applications ◽  
Binding Cleft ◽  
Structural Insights ◽  
Hydrolase Family

β-Mannosidases are exo-acting glycoside hydrolases (GHs) that catalyse the removal of the nonreducing end β-D-mannose from manno-oligosaccharides or mannoside-substituted molecules. They play important roles in fundamental biological processes and also have potential applications in various industries. In this study, the first fungal GH family 5 β-mannosidase (RmMan5B) fromRhizomucor mieheiwas functionally and structurally characterized.RmMan5B exhibited a much higher activity against manno-oligosaccharides than againstp-nitrophenyl β-D-mannopyranoside (pNPM) and had a transglycosylation activity which transferred mannosyl residues to sugars such as fructose. To investigate its substrate specificity and transglycosylation activity, crystal structures ofRmMan5B and of its inactive E202A mutant in complex with mannobiose, mannotriose and mannosyl-fructose were determined at resolutions of 1.3, 2.6, 2.0 and 2.4 Å, respectively. In addition, the crystal structure ofR. mieheiβ-mannanase (RmMan5A) was determined at a resolution of 2.3 Å. BothRmMan5A andRmMan5B adopt the (β/α)8-barrel architecture, which is globally similar to the other members of GH family 5. However,RmMan5B shows several differences in the loop around the active site. The extended loop between strand β8 and helix α8 (residues 354–392) forms a `double' steric barrier to `block' the substrate-binding cleft at the end of the −1 subsite. Trp119, Asn260 and Glu380 in the β-mannosidase, which are involved in hydrogen-bond contacts with the −1 mannose, might be essential for exo catalytic activity. Moreover, the structure of RmMan5B in complex with mannosyl-fructose has provided evidence for the interactions between the β-mannosidase and D-fructofuranose. Overall, the present study not only helps in understanding the catalytic mechanism of GH family 5 β-mannosidases, but also provides a basis for further enzymatic engineering of β-mannosidases and β-mannanases.

scholarly journals Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)

2018 ◽  
Vol 293 (47) ◽  
pp. 18138-18150 ◽  
Author(s):  
Léa Chuzel ◽  
Mehul B. Ganatra ◽  
Erdmann Rapp ◽  
Bernard Henrissat ◽  
Christopher H. Taron
Keyword(s):  
Glycoside Hydrolase ◽  
Catalytic Mechanism ◽  
Recombinant Expression ◽  
Biochemical Property ◽  
Environmental Dna ◽  
Thermal Spring ◽  
Sialic Acid Residue ◽  
Glycoside Hydrolases ◽  
Glycoside Hydrolase Family ◽  
Hydrolase Family

Exosialidases are glycoside hydrolases that remove a single terminal sialic acid residue from oligosaccharides. They are widely distributed in biology, having been found in prokaryotes, eukaryotes, and certain viruses. Most characterized prokaryotic sialidases are from organisms that are pathogenic or commensal with mammals. However, in this study, we used functional metagenomic screening to seek microbial sialidases encoded by environmental DNA isolated from an extreme ecological niche, a thermal spring. Using recombinant expression of potential exosialidase candidates and a fluorogenic sialidase substrate, we discovered an exosialidase having no homology to known sialidases. Phylogenetic analysis indicated that this protein is a member of a small family of bacterial proteins of previously unknown function. Proton NMR revealed that this enzyme functions via an inverting catalytic mechanism, a biochemical property that is distinct from those of known exosialidases. This unique inverting exosialidase defines a new CAZy glycoside hydrolase family we have designated GH156.

scholarly journals Epoxyalkyl glycosides of d-xylose and xylo-oligosaccharides are active-site markers of xylanases from glycoside hydrolase family 11, not from family 10

2000 ◽  
Vol 347 (3) ◽  
pp. 865-873 ◽  
Author(s):  
Patricia NTARIMA ◽  
Wim NERINCKX ◽  
Klaus KLARSKOV ◽  
Bart DEVREESE ◽  
Mahalingeshwara K. BHAT ◽  
...  
Keyword(s):  
Active Site ◽  
Glycoside Hydrolase ◽  
Glycoside Hydrolases ◽  
Thermomyces Lanuginosus ◽  
Glycoside Hydrolase Family ◽  
Active Site Residues ◽  
X Ray ◽  
Order Of Magnitude ◽  
Site Markers ◽  
Hydrolase Family

A series of Ω-epoxyalkyl glycosides of D-xylopyranose, xylobiose and xylotriose were tested as potential active-site-directed inhibitors of xylanases from glycoside hydrolase families 10 and 11. Whereas family-10 enzymes (Thermoascus aurantiacus Xyn and Clostridium thermocellum Xyn Z) are resistant to electrophilic attack of active-site carboxyl residues, glycoside hydrolases of family 11 (Thermomyces lanuginosus Xyn and Trichoderma reesei Xyn II) are irreversibly inhibited. The apparent inactivation and association constants (ki, 1/Ki) are one order of magnitude higher for the xylobiose and xylotriose derivatives. The effects of the aglycone chain length can clearly be described. Xylobiose and n-alkyl β-D-xylopyranosides are competitive ligands and provide protection against inactivation. MS measurements showed 1:1 stoichiometries in most labelling experiments. Electrospray ionization MS/MS analysis revealed the nucleophile Glu86 as the modified residue in the T. lanuginosus xylanase when 2,3-epoxypropyl β-D-xylopyranoside was used, whereas the acid/base catalyst Glu178 was modified by the 3,4-epoxybutyl derivative. The active-site residues Glu86 and Glu177 in T. reesei Xyn II are similarly modified, confirming earlier X-ray crystallographic data [Havukainen, Törrönen, Laitinen and Rouvinen (1996) Biochemistry 35, 9617-9624]. The inability of the Ω-epoxyalkyl xylo(oligo)saccharide derivatives to inactivate family-10 enzymes is discussed in terms of different ligand-subsite interactions.

scholarly journals Characterization of Glycoside Hydrolase Families 13 and 31 Reveals Expansion and Diversification of α-Amylase Genes in the Phlebotomine Lutzomyia longipalpis and Modulation of Sandfly Glycosidase Activities by Leishmania Infection

2021 ◽  
Vol 12 ◽  
Author(s):  
Samara Graciane da Costa-Latgé ◽  
Paul Bates ◽  
Rod Dillon ◽  
Fernando Ariel Genta
Keyword(s):  
Glycoside Hydrolase ◽  
The Body ◽  
Glycoside Hydrolases ◽  
Food Sources ◽  
Leishmania Infection ◽  
Glycoside Hydrolase Family ◽  
Lutzomyia Longipalpis ◽  
Leishmania Mexicana ◽  
Hydrolase Family ◽  
Dipteran Species

Sugar-rich food sources are essential for sandflies to meet their energy demands, achieving more prolonged survival. The digestion of carbohydrates from food is mainly realized by glycoside hydrolases (GH). To identify genes coding for α-glycosidases and α-amylases belonging to Glycoside Hydrolase Family 13 (GH13) and Glycoside Hydrolase Family 31 (GH31) in Lutzomyia longipalpis, we performed an HMMER search against its genome using known sequences from other dipteran species. The sequences retrieved were classified based on BLASTP best hit, analysis of conserved regions by alignment with sequences of proteins with known structure, and phylogenetic analysis comparing with orthologous proteins from other dipteran species. Using RT-PCR analysis, we evaluated the expression of GH13 and GH31 genes, in the gut and rest of the body of females, in four different conditions: non-fed, sugar-fed, blood-fed, and Leishmania mexicana infected females. L. longipalpis has GH13/31 genes that code for enzymes involved in various aspects of sugar metabolism, as carbohydrate digestion, storage, and mobilization of glycogen reserves, proteins involved in transport, control of N-glycosylation quality, as well as others with a putative function in the regulation of myogenesis. These proteins are representatives of GH13 and GH31 families, and their roles seem to be conserved. Most of the enzymes seem to be active with conserved consense sequences, including the expected catalytic residues. α-amylases also demonstrated the presence of calcium and chloride binding sites. L. longipalpis genome shows an expansion in the α-amylase gene family, with two clusters. In contrast, a retraction in the number of α-glucosidases occurred. The expansion of α-amylases is probably related to the specialization of these proteins for different substrates or inhibitors, which might correlate with the higher diversity of plant foods available in the natural habitat of L. longipalpis. The expression of α-glucosidase genes is higher in blood-fed females, suggesting their role in blood digestion. Besides that, in blood-fed females infected with the parasite Leishmania mexicana, these genes were also modulated. Glycoside Hydrolases from families 13 and 31 are essential for the metabolism of L. longipalpis, and GH13 enzymes seem to be involved in the interaction between sandflies and Leishmania.

scholarly journals Functional analysis of a group A streptococcal glycoside hydrolase Spy1600 from family 84 reveals it is a β-N-acetylglucosaminidase and not a hyaluronidase

2006 ◽  
Vol 399 (2) ◽  
pp. 241-247 ◽  
Author(s):  
William L. Sheldon ◽  
Matthew S. Macauley ◽  
Edward J. Taylor ◽  
Charlotte E. Robinson ◽  
Simon J. Charnock ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Catalytic Mechanism ◽  
Group A Streptococcus ◽  
Competitive Inhibitor ◽  
Glycoside Hydrolases ◽  
Glycoside Hydrolase Family ◽  
Toxic Shock ◽  
Nmr Studies ◽  
Invasive Infections ◽  
Group A

Group A streptococcus (Streptococcus pyogenes) is the causative agent of severe invasive infections such as necrotizing fasciitis (the so-called ‘flesh eating disease’) and toxic-shock syndrome. Spy1600, a glycoside hydrolase from family 84 of the large superfamily of glycoside hydrolases, has been proposed to be a virulence factor. In the present study we show that Spy1600 has no activity toward galactosaminides or hyaluronan, but does remove β-O-linked N-acetylglucosamine from mammalian glycoproteins – an observation consistent with the inclusion of eukaryotic O-glycoprotein 2-acetamido-2-deoxy-β-D-glucopyranosidases within glycoside hydrolase family 84. Proton NMR studies, structure–reactivity studies for a series of fluorinated analogues and analysis of 1,2-dideoxy-2′-methyl-α-D-glucopyranoso-[2,1-d]-Δ2′-thiazoline as a competitive inhibitor reveals that Spy1600 uses a double-displacement mechanism involving substrate-assisted catalysis. Family 84 glycoside hydrolases are therefore comprised of both prokaryotic and eukaryotic β-N-acetylglucosaminidases using a conserved catalytic mechanism involving substrate-assisted catalysis. Since these enzymes do not have detectable hyaluronidase activity, many family 84 glycoside hydrolases are most likely incorrectly annotated as hyaluronidases.

scholarly journals Two exo-β-D-glucosaminidases/exochitosanases from actinomycetes define a new subfamily within family 2 of glycoside hydrolases

2006 ◽  
Vol 394 (3) ◽  
pp. 675-686 ◽  
Author(s):  
Nathalie Côté ◽  
Alain Fleury ◽  
Émilie Dumont-Blanchette ◽  
Tamo Fukamizo ◽  
Masaru Mitsutomi ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Catalytic Mechanism ◽  
Pcr Primers ◽  
Amino Acid Sequences ◽  
Glycoside Hydrolases ◽  
Cloning And Expression ◽  
Streptomyces Avermitilis ◽  
Gene Cloning And Expression ◽  
Glycoside Hydrolase Family ◽  
Sequence Alignments

A GlcNase (exo-β-D-glucosaminidase) was purified from culture supernatant of Amycolatopsis orientalis subsp. orientalis grown in medium with chitosan. The enzyme hydrolysed the terminal GlcN (glucosamine) residues in oligomers of GlcN with transglycosylation observed at late reaction stages. 1H-NMR spectroscopy revealed that the enzyme is a retaining glycoside hydrolase. The GlcNase also behaved as an exochitosanase against high-molecular-mass chitosan with Km and kcat values of 0.16 mg/ml and 2832 min−1. On the basis of partial amino acid sequences, PCR primers were designed and used to amplify a DNA fragment which then allowed the cloning of the GlcNase gene (csxA) associated with an open reading frame of 1032 residues. The GlcNase has been classified as a member of glycoside hydrolase family 2 (GH2). Sequence alignments identified a group of CsxA-related protein sequences forming a distinct GH2 subfamily. Most of them have been annotated in databases as putative β-mannosidases. Among these, the SAV1223 protein from Streptomyces avermitilis has been purified following gene cloning and expression in a heterologous host and shown to be a GlcNase with no detectable β-mannosidase activity. In CsxA and all relatives, a serine-aspartate doublet replaces an asparagine residue and a glutamate residue, which were strictly conserved in previously studied GH2 members with β-galactosidase, β-glucuronidase or β-mannosidase activity and shown to be directly involved in various steps of the catalytic mechanism. Alignments of several other GH2 members allowed the identification of yet another putative subfamily, characterized by a novel, serine-glutamate doublet at these positions.

scholarly journals Crystal Structure of α-1,4-Glucan Lyase, a Unique Glycoside Hydrolase Family Member with a Novel Catalytic Mechanism

2013 ◽  
Vol 288 (37) ◽  
pp. 26764-26774 ◽  
Author(s):  
Henriëtte J. Rozeboom ◽  
Shukun Yu ◽  
Susan Madrid ◽  
Kor H. Kalk ◽  
Ran Zhang ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Family Member ◽  
Glycoside Hydrolase ◽  
Catalytic Mechanism ◽  
Glycoside Hydrolase Family ◽  
Hydrolase Family

scholarly journals A novel β-xylosidase structure fromGeobacillus thermoglucosidasius: the first crystal structure of a glycoside hydrolase family GH52 enzyme reveals unpredicted similarity to other glycoside hydrolase folds

2014 ◽  
Vol 70 (5) ◽  
pp. 1366-1374 ◽  
Author(s):  
Giannina Espina ◽  
Kirstin Eley ◽  
Guillaume Pompidor ◽  
Thomas R. Schneider ◽  
Susan J. Crennell ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Glycoside Hydrolase ◽  
Thermophilic Bacterium ◽  
Glycoside Hydrolases ◽  
Glycoside Hydrolase Family ◽  
Gene Encoding ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
The Family ◽  
Hydrolase Family

Geobacillus thermoglucosidasiusis a thermophilic bacterium that is able to ferment both C6 and C5 sugars to produce ethanol. During growth on hemicellulose biomass, an intracellular β-xylosidase catalyses the hydrolysis of xylo-oligosaccharides to the monosaccharide xylose, which can then enter the pathways of central metabolism. The gene encoding aG. thermoglucosidasiusβ-xylosidase belonging to CAZy glycoside hydrolase family GH52 has been cloned and expressed inEscherichia coli. The recombinant enzyme has been characterized and a high-resolution (1.7 Å) crystal structure has been determined, resulting in the first reported structure of a GH52 family member. A lower resolution (2.6 Å) structure of the enzyme–substrate complex shows the positioning of the xylobiose substrate to be consistent with the proposed retaining mechanism of the family; additionally, the deep cleft of the active-site pocket, plus the proximity of the neighbouring subunit, afford an explanation for the lack of catalytic activity towards the polymer xylan. Whilst the fold of theG. thermoglucosidasiusβ-xylosidase is completely different from xylosidases in other CAZy families, the enzyme surprisingly shares structural similarities with other glycoside hydrolases, despite having no more than 13% sequence identity.

scholarly journals Two distinct catalytic pathways for GH43 xylanolytic enzymes unveiled by X-ray and QM/MM simulations

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Mariana A. B. Morais ◽  
Joan Coines ◽  
Mariane N. Domingues ◽  
Renan A. S. Pirolla ◽  
Celisa C. C. Tonoli ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
Open Architecture ◽  
Reaction Pathways ◽  
Main Reaction ◽  
Glycoside Hydrolase Family ◽  
X Ray ◽  
Glycoside Hydrolase Family 43 ◽  
Xylanolytic Enzymes ◽  
Hydrolase Family

AbstractXylanolytic enzymes from glycoside hydrolase family 43 (GH43) are involved in the breakdown of hemicellulose, the second most abundant carbohydrate in plants. Here, we kinetically and mechanistically describe the non-reducing-end xylose-releasing exo-oligoxylanase activity and report the crystal structure of a native GH43 Michaelis complex with its substrate prior to hydrolysis. Two distinct calcium-stabilized conformations of the active site xylosyl unit are found, suggesting two alternative catalytic routes. These results are confirmed by QM/MM simulations that unveil the complete hydrolysis mechanism and identify two possible reaction pathways, involving different transition state conformations for the cleavage of xylooligosaccharides. Such catalytic conformational promiscuity in glycosidases is related to the open architecture of the active site and thus might be extended to other exo-acting enzymes. These findings expand the current general model of catalytic mechanism of glycosidases, a main reaction in nature, and impact on our understanding about their interaction with substrates and inhibitors.

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