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.

scholarly journals The structure of a family GH25 lysozyme fromAspergillus fumigatus

2010 ◽  
Vol 66 (9) ◽  
pp. 973-977 ◽  
Author(s):  
Justyna E. Korczynska ◽  
Steffen Danielsen ◽  
Ulrika Schagerlöf ◽  
Johan P. Turkenburg ◽  
Gideon J. Davies ◽  
...  
Keyword(s):  
Active Site ◽  
Glycoside Hydrolase ◽  
Catalytic Mechanism ◽  
Medical Application ◽  
Glycoside Hydrolase Family ◽  
X Ray ◽  
Fungal Structure ◽  
The Family ◽  
Hydrolase Family ◽  
Insight Into

Lysins are important biomolecules which cleave the bacterial cell-wall polymer peptidoglycan. They are finding increasing commercial and medical application. In order to gain an insight into the mechanism by which these enzymes operate, the X-ray structure of a CAZy family GH25 `lysozyme' fromAspergillus fumigatuswas determined. This is the first fungal structure from the family and reveals a modified α/β-barrel-like fold in which an eight-stranded β-barrel is flanked by three α-helices. The active site lies toward the bottom of a negatively charged pocket and its layout has much in common with other solved members of the GH25 and related GH families. A conserved active-site DXE motif may be implicated in catalysis, lending further weight to the argument that this glycoside hydrolase family operatesviaa `substrate-assisted' catalytic mechanism.

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.

A Novel Multifunctional Arabinofuranosidase/Endo-xylanase/β-Xylosidase GH43 from Paenibacillus curdlanolyticus B-6 and Its Synergistic Action to Produce Arabinose and Xylose from Cereal Arabinoxylan

2021 ◽  
Author(s):  
Puangpen Limsakul ◽  
Paripok Phitsuwan ◽  
Rattiya Waeonukul ◽  
Patthra Pason ◽  
Chakrit Tachaapaikoon ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Synergistic Action ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Glycoside Hydrolase Family 43 ◽  
Paenibacillus Curdlanolyticus ◽  
Xylanolytic Enzyme ◽  
Almost All ◽  
Xylanolytic Enzymes ◽  
Hydrolase Family

The PcAxy43B is a modular protein comprising a catalytic domain of glycoside hydrolase family 43 (GH43), a family 6 carbohydrate-binding module (CBM6) and a family 36 carbohydrate-binding module (CBM36) and found to be a novel multifunctional xylanolytic enzyme from Paenibacillus curdlanolyticus B-6. This enzyme exhibited α-L-arabinofuranosidase, endo-xylanase and β-D-xylosidase activities. α-L-Arabinofuranosidase of PcAxy43B revealed the new property of GH43, which released arabinose from the short-chain arabinoxylo-oligosaccharide (AXOS) and cereal arabinoxylan, and from both sides of the xylose residues of AXOS, which usually obstruct the action of xylanolytic enzymes. The PcAxy43B liberated series of xylo-oligosaccharides (XOSs) from birchwood xylan and xylohexaose, indicating that PcAxy43B exhibited endo-xylanase activity. The PcAxy43B produced xylose from xylobiose and reacted with p -nitrophenyl-β-D-xylopyranoside as a result of β-xylosidase activity. The PcAxy43B effectively released arabinose together with XOSs and xylose from the highly arabinosyl-substituted rye arabinoxylan. Moreover, PcAxy43B showed significant synergistic action with a trifunctional endo-xylanase/β-xylosidase/α-L-arabinofuranosidase PcAxy43A and an endo-xylanase Xyn10C from the strain B-6, in which almost all products produced from rye arabinoxylan by these combined enzymes were arabinose and xylose. In addition, the presence of CBM36 was found to be necessary for the endo-xylanase property of PcAxy43B. The PcAxy43B is capable of hydrolysing untreated cereal biomass, corn hull and rice straw into XOSs and xylose. Hence, PcAxy43B, the significant accessory multifunctional xylanolytic enzyme, is a potential candidate for application in the saccharification of cereal biomass. IMPORTANCE Enzymatic saccharification of cereal biomass is a strategy for the production of fermented sugars from low-price raw materials. In the present study, PcAxy43B from P. curdlanolyticus B-6 was found to be a novel multifunctional α-L-arabinofuranosidase/endo-xylanase/β-D-xylosidase enzyme of the glycoside hydrolase family 43. It is effective in releasing arabinose, xylose and XOSs from the highly arabinosyl-substituted rye arabinoxylan, which is usually resistant to hydrolysis by xylanolytic enzymes. Moreover, almost all products produced from rye arabinoxylan by the combination of PcAxy43B with trifunctional xylanolytic enzyme PcAxy43A and endo-xylanase Xyn10C from the strain B-6 were arabinose and xylose, which can be used to produce several value-added products. In addition, PcAxy43B is capable of hydrolysing untreated cereal biomass into XOSs and xylose. Thus, PcAxy43B is an important multifunctional xylanolytic enzyme with high potential in biotechnology.

scholarly journals Biochemical characterization of a glycoside hydrolase family 43 β-D-galactofuranosidase from the fungus Aspergillus niger

2021 ◽  
Author(s):  
Gregory S Bulmer ◽  
Fang Wei Yuen ◽  
Naimah Begum ◽  
Bethan S Jones ◽  
Sabine S Flitsch ◽  
...  
Keyword(s):  
Aspergillus Niger ◽  
Glycoside Hydrolase ◽  
Biochemical Characterization ◽  
Maximum Activity ◽  
Glycoside Hydrolase Family ◽  
Enzyme Specificity ◽  
Fungal Enzymes ◽  
Glycoside Hydrolase Family 43 ◽  
Hydrolase Family

β-D-Galactofuranose (Galf) and its polysaccharides are found in bacteria, fungi and protozoa but do not occur in mammalian tissues, and thus represent a specific target for anti-pathogenic drugs. Understanding the enzymatic degradation of these polysaccharides is therefore of great interest, but the identity of fungal enzymes with exclusively galactofuranosidase activity has so far remained elusive. Here we describe the identification and characterization of a galactofuranosidase from the industrially important fungus Aspergillus niger. Phylogenetic analysis of glycoside hydrolase family 43 subfamily 34 (GH43_34) members revealed the occurrence of three distinct clusters and, by comparison with specificities of characterized bacterial members, suggested a basis for prediction of enzyme specificity. Using this rationale, in tandem with molecular docking, we identified a putative β-D-galactofuranosidase from A. niger which was recombinantly expressed in Escherichia coli. The Galf-specific hydrolase, encoded by xynD demonstrates maximum activity at pH 5, 25 °C towards 4-Nitrophenyl-β-galactofuranoside (pNP-βGalf), with a Km of 17.9 ± 1.9 mM and Vmax of 70.6 ± 5.3 μmol min-1. The characterization of this first fungal GH43 galactofuranosidase offers further molecular insight into the degradation of Galf-containing structures and may inform clinical treatments against fungal pathogens.

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.

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