Active-pocket size differentiating insectile from bacterial chitinolytic β-N-acetyl-D-hexosaminidases

2011 ◽  
Vol 438 (3) ◽  
pp. 467-474 ◽  
Author(s):  
Tian Liu ◽  
Haitao Zhang ◽  
Fengyi Liu ◽  
Lei Chen ◽  
Xu Shen ◽  
...  
Keyword(s):  
Active Site ◽  
Glycoside Hydrolase Family ◽  
Complex Structures ◽  
Ostrinia Furnacalis ◽  
Wild Type ◽  
Structural Differences ◽  
Streptomyces Plicatus ◽  
Species Specific ◽  
Micro Organisms ◽  
Hydrolase Family

Chitinolytic β-N-acetyl-D-hexosaminidase is a branch of the GH20 (glycoside hydrolase family 20) β-N-acetyl-D-hexosaminidases that is only distributed in insects and micro-organisms, and is therefore a potential target for the action of insecticides. PUGNAc [O-(2-acetamido-2-deoxy-D-glucopyransylidene)-amino-N-phenylcarbamate] was initially identified as an inhibitor against GH20 β-N-acetyl-D-hexosaminidases. So far no crystal structure of PUGNAc in complex with any GH20 β-N-acetyl-D-hexosaminidase has been reported. We show in the present study that the sensitivities of chitinolytic β-N-acetyl-D-hexosaminidases towards PUGNAc can vary by 100-fold, with the order being OfHex1 (Ostrinia furnacalis β-N-acetyl-D-hexosaminidase)<SmCHB (Serratia marcescens chitobiase)<SpHex (Streptomyces plicatus β-N-acetyl-D-hexosaminidase). To explain this difference, the crystal structures of wild-type OfHex1 as well as mutant OfHex1(V327G) in complex with PUGNAc were determined at 2.0 Å (1 Å=0.1 nm) and 2.3 Å resolutions and aligned with the complex structures of SpHex and SmCHB. The results showed that the sensitivities of these enzymes to PUGNAc were determined by the active pocket size, with OfHex1 having the largest but narrowest entrance, whereas SpHex has the smallest entrance, suitable for holding the inhibitor, and SmCHB has the widest entrance. By widening the size of the active pocket entrance of OfHex1 through replacing the active site Val327 with a glycine residue, the sensitivity of OfHex1 to PUGNAc became similar to that of SmCHB. The structural differences among chitinolytic β-N-acetyl-D-hexosaminidases leading to different sensitivities to PUGNAc may be useful for developing species-specific pesticides and bactericides.

scholarly journals Active Site and Laminarin Binding in Glycoside Hydrolase Family 55

2015 ◽  
Vol 290 (19) ◽  
pp. 11819-11832 ◽  
Author(s):  
Christopher M. Bianchetti ◽  
Taichi E. Takasuka ◽  
Sam Deutsch ◽  
Hannah S. Udell ◽  
Eric J. Yik ◽  
...  
Keyword(s):  
Active Site ◽  
Glycoside Hydrolase ◽  
Glycoside Hydrolase Family ◽  
Hydrolase Family

Pseudomonas cellulosa expresses a single membrane-bound glycoside hydrolase family 51 arabinofuranosidase

2001 ◽  
Vol 358 (3) ◽  
pp. 599-605 ◽  
Author(s):  
Marie-Helene BEYLOT ◽  
Kaveh EMAMI ◽  
Vincent A. McKIE ◽  
Harry J. GILBERT ◽  
Gavin PELL
Keyword(s):  
Sugar Beet ◽  
Glycoside Hydrolase ◽  
Northern Blot Analysis ◽  
Glycoside Hydrolase Family ◽  
Wild Type ◽  
Mutant Bacterium ◽  
Membrane Bound ◽  
Hydrolase Family ◽  
Wild Type Bacterium ◽  
Glycoside Hydrolase Family 51

In the accompanying paper [Beylot, McKie, Voragen, Doeswijk-Voragen and Gilbert (2001) Biochem. J. 358, 607–614] the chromosome of Pseudomonas cellulosa was shown to contain two genes, abf51A and abf62A, that encode arabinofuranosidases belonging to glycoside hydrolase families 51 and 62, respectively. In this report we show that expression of Abf51A is induced by arabinose and arabinose-containing polysaccharides. Northern-blot analysis showed that abf51A was efficiently transcribed, whereas no transcript derived from abf62A was detected in the presence of arabinose-containing polysaccharides. Zymogram and Western-blot analyses revealed that Abf51A was located on the outer membrane of P. cellulosa. To investigate the importance of Abf51A in the release of arabinose from poly- and oligosaccharides, transposon mutagenesis was used to construct an abf51A-inactive mutant of P. cellulosa (Δabf51A). The mutant did not grow on linear arabinan or sugar beet arabinan, and utilized arabinoxylan much more slowly than the wild-type bacterium. Arabinofuranosidase activity in Δabf51A against aryl-α-arabinofuranosides, arabinan and α1,5-linked arabino-oligosaccharides was approx. 1% of the wild-type bacterium. The mutant bacterium did not exhibit arabinofuranosidase activity against arabinoxylan, supporting the view that abf62A is not expressed in P. cellulosa. These data indicate that P. cellulosa expresses a membrane-bound glycoside hydrolase family 51 arabinofuranosidase that plays a pivotal role in releasing arabinose from polysaccharides and arabino-oligosaccharides.

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.

Crystal structures of the GH6 Orpinomyces sp. Y102 CelC7 enzyme with exo and endo activity and its complex with cellobiose

2019 ◽  
Vol 75 (12) ◽  
pp. 1138-1147
Author(s):  
Hsiao-Chuan Huang ◽  
Liu-Hong Qi ◽  
Yo-Chia Chen ◽  
Li-Chu Tsai
Keyword(s):  
Enzymatic Hydrolysis ◽  
Crystal Structures ◽  
Active Site ◽  
Binding Sites ◽  
Glycoside Hydrolase ◽  
Catalytic Domain ◽  
Glycoside Hydrolase Family ◽  
Substrate Binding Sites ◽  
Key Residues ◽  
Hydrolase Family

The catalytic domain (residues 128–449) of the Orpinomyces sp. Y102 CelC7 enzyme (Orp CelC7) exhibits cellobiohydrolase and cellotriohydrolase activities. Crystal structures of Orp CelC7 and its cellobiose-bound complex have been solved at resolutions of 1.80 and 2.78 Å, respectively. Cellobiose occupies subsites +1 and +2 within the active site of Orp CelC7 and forms hydrogen bonds to two key residues: Asp248 and Asp409. Furthermore, its substrate-binding sites have both tunnel-like and open-cleft conformations, suggesting that the glycoside hydrolase family 6 (GH6) Orp CelC7 enzyme may perform enzymatic hydrolysis in the same way as endoglucanases and cellobiohydrolases. LC-MS/MS analysis revealed cellobiose (major) and cellotriose (minor) to be the respective products of endo and exo activity of the GH6 Orp CelC7.

Study of the Active Site Residues of a Glycoside Hydrolase Family 8 Xylanase

2005 ◽  
Vol 354 (2) ◽  
pp. 425-435 ◽  
Author(s):  
T. Collins ◽  
D. De Vos ◽  
A. Hoyoux ◽  
S.N. Savvides ◽  
C. Gerday ◽  
...  
Keyword(s):  
Active Site ◽  
Glycoside Hydrolase ◽  
Glycoside Hydrolase Family ◽  
Active Site Residues ◽  
Hydrolase Family ◽  
Glycoside Hydrolase Family 8

scholarly journals Crystal Structure of Cel44A, a Glycoside Hydrolase Family 44 Endoglucanase from Clostridium thermocellum

2007 ◽  
Vol 282 (49) ◽  
pp. 35703-35711 ◽  
Author(s):  
Yu Kitago ◽  
Shuichi Karita ◽  
Nobuhisa Watanabe ◽  
Masakatsu Kamiya ◽  
Tomoyasu Aizawa ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Glutamic Acid ◽  
Glycoside Hydrolase ◽  
Clostridium Thermocellum ◽  
Structural Features ◽  
Glycoside Hydrolase Family ◽  
Wild Type ◽  
H Nmr ◽  
Hydrolase Family

The crystal structure of Cel44A, which is one of the enzymatic components of the cellulosome of Clostridium thermocellum, was solved at a resolution of 0.96Å. This enzyme belongs to glycoside hydrolase family (GH family) 44. The structure reveals that Cel44A consists of a TIM-like barrel domain and a β-sandwich domain. The wild-type and the E186Q mutant structures complexed with substrates suggest that two glutamic acid residues, Glu186 and Glu359, are the active residues of the enzyme. Biochemical experiments were performed to confirm this idea. The structural features indicate that GH family 44 belongs to clan GH-A and that the reaction catalyzed by Cel44A is retaining type hydrolysis. The stereochemical course of hydrolysis was confirmed by a 1H NMR experiment using the reduced cellooligosaccharide as a substrate.

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 Redirecting substrate regioselectivity using engineered ΔN123-GBD-CD2 branching sucrases for the production of pentasaccharide repeating units of S. flexneri 3a, 4a and 4b haptens

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Mounir Benkoulouche ◽  
Akli Ben Imeddourene ◽  
Louis-Antoine Barel ◽  
Guillaume Le Heiget ◽  
Sandra Pizzut ◽  
...  
Keyword(s):  
Active Site ◽  
Shigella Flexneri ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Active Enzymes ◽  
Enzyme Active Site ◽  
Molecular Determinants ◽  
Dynamics Simulations ◽  
Carbohydrate Components ◽  
Glycosylation Reaction ◽  
Hydrolase Family

AbstractThe (chemo-)enzymatic synthesis of oligosaccharides has been hampered by the lack of appropriate enzymatic tools with requisite regio- and stereo-specificities. Engineering of carbohydrate-active enzymes, in particular targeting the enzyme active site, has notably led to catalysts with altered regioselectivity of the glycosylation reaction thereby enabling to extend the repertoire of enzymes for carbohydrate synthesis. Using a collection of 22 mutants of ΔN123-GBD-CD2 branching sucrase, an enzyme from the Glycoside Hydrolase family 70, containing between one and three mutations in the active site, and a lightly protected chemically synthesized tetrasaccharide as an acceptor substrate, we showed that altered glycosylation product specificities could be achieved compared to the parental enzyme. Six mutants were selected for further characterization as they produce higher amounts of two favored pentasaccharides compared to the parental enzyme and/or new products. The produced pentasaccharides were shown to be of high interest as they are precursors of representative haptens of Shigella flexneri serotypes 3a, 4a and 4b. Furthermore, their synthesis was shown to be controlled by the mutations introduced in the active site, driving the glucosylation toward one extremity or the other of the tetrasaccharide acceptor. To identify the molecular determinants involved in the change of ΔN123-GBD-CD2 regioselectivity, extensive molecular dynamics simulations were carried out in combination with in-depth analyses of amino acid residue networks. Our findings help to understand the inter-relationships between the enzyme structure, conformational flexibility and activity. They also provide new insight to further engineer this class of enzymes for the synthesis of carbohydrate components of bacterial haptens.

scholarly journals Structural basis of catalysis and substrate recognition by the NAD(H)-dependent α-d-glucuronidase from the glycoside hydrolase family 4

2021 ◽  
Vol 478 (4) ◽  
pp. 943-959
Author(s):  
Samar Ballabha Mohapatra ◽  
Narayanan Manoj
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Glycoside Hydrolase ◽  
Metal Ion ◽  
Substrate Recognition ◽  
Structural Basis ◽  
Glycoside Hydrolase Family ◽  
Diverse Range ◽  
Molecular Features ◽  
Hydrolase Family

Members of the glycoside hydrolase family 4 (GH4) employ an unusual glycosidic bond cleavage mechanism utilizing NAD(H) and a divalent metal ion, under reducing conditions. These enzymes act upon a diverse range of glycosides, and unlike most other GH families, homologs here are known to accommodate both α- and β-anomeric specificities within the same active site. Here, we report the catalytic properties and the crystal structures of TmAgu4B, an α-d-glucuronidase from the hyperthermophile Thermotoga maritima. The structures in three different states include the apo form, the NADH bound holo form, and the ternary complex with NADH and the reaction product d-glucuronic acid, at 2.15, 1.97 and 1.85 Å resolutions, respectively. These structures reveal the step-wise route of conformational changes required in the active site to achieve the catalytically competent state, and illustrate the direct role of residues that determine the reaction mechanism. Furthermore, a structural transition of a helical region in the active site to a turn geometry resulting in the rearrangement of a unique arginine residue governs the exclusive glucopyranosiduronic acid recognition in TmAgu4B. Mutational studies show that modifications of the glycone binding site geometry lead to catalytic failure and indicate overlapping roles of specific residues in catalysis and substrate recognition. The data highlight hitherto unreported molecular features and associated active site dynamics that determine the structure–function relationships within the unique GH4 family.

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