scholarly journals Crystallographic analysis shows substrate binding at the −3 to +1 active-site subsites and at the surface of glycoside hydrolase family 11 endo-1,4-β-xylanases

2008 ◽  
Vol 410 (1) ◽  
pp. 71-79 ◽  
Author(s):  
Elien Vandermarliere ◽  
Tine M. Bourgois ◽  
Sigrid Rombouts ◽  
Steven van Campenhout ◽  
Guido Volckaert ◽  
...  
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Glycoside Hydrolase ◽  
Plant Cell Wall ◽  
Substrate Binding ◽  
Crystallographic Analysis ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Modules ◽  
Hydrolase Family

GH 11 (glycoside hydrolase family 11) xylanases are predominant enzymes in the hydrolysis of heteroxylan, an abundant structural polysaccharide in the plant cell wall. To gain more insight into the protein–ligand interactions of the glycone as well as the aglycone subsites of these enzymes, catalytically incompetent mutants of the Bacillus subtilis and Aspergillus niger xylanases were crystallized, soaked with xylo-oligosaccharides and subjected to X-ray analysis. For both xylanases, there was clear density for xylose residues in the −1 and −2 subsites. In addition, for the B. subtilis xylanase, there was also density for xylose residues in the −3 and +1 subsite showing the spanning of the −1/+1 subsites. These results, together with the observation that some residues in the aglycone subsites clearly adopt a different conformation upon substrate binding, allowed us to identify the residues important for substrate binding in the aglycone subsites. In addition to substrate binding in the active site of the enzymes, the existence of an unproductive second ligand-binding site located on the surface of both the B. subtilis and A. niger xylanases was observed. This extra binding site may have a function similar to the separate carbohydrate-binding modules of other glycoside hydrolase families.

scholarly journals Structural analysis of a glycoside hydrolase family 43 arabinoxylan arabinofuranohydrolase in complex with xylotetraose reveals a different binding mechanism compared with other members of the same family

2009 ◽  
Vol 418 (1) ◽  
pp. 39-47 ◽  
Author(s):  
Elien Vandermarliere ◽  
Tine M. Bourgois ◽  
Martyn D. Winn ◽  
Steven van Campenhout ◽  
Guido Volckaert ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Catalytic Domain ◽  
Crystallographic Analysis ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Stacking Interactions ◽  
Glycoside Hydrolase Family 43 ◽  
Arabinoxylan Arabinofuranohydrolase ◽  
Hydrolase Family

AXHs (arabinoxylan arabinofuranohydrolases) are α-L-arabinofuranosidases that specifically hydrolyse the glycosidic bond between arabinofuranosyl substituents and xylopyranosyl backbone residues of arabinoxylan. Bacillus subtilis was recently shown to produce an AXH that cleaves arabinose units from O-2- or O-3-mono-substituted xylose residues: BsAXH-m2,3 (B. subtilis AXH-m2,3). Crystallographic analysis reveals a two-domain structure for this enzyme: a catalytic domain displaying a five-bladed β-propeller fold characteristic of GH (glycoside hydrolase) family 43 and a CBM (carbohydrate-binding module) with a β-sandwich fold belonging to CBM family 6. Binding of substrate to BsAXH-m2,3 is largely based on hydrophobic stacking interactions, which probably allow the positional flexibility needed to hydrolyse both arabinose substituents at the O-2 or O-3 position of the xylose unit. Superposition of the BsAXH-m2,3 structure with known structures of the GH family 43 exo-acting enzymes, β-xylosidase and α-L-arabinanase, each in complex with their substrate, reveals a different orientation of the sugar backbone.

scholarly journals CalkGH9T: A Glycoside Hydrolase Family 9 Enzyme from Clostridium alkalicellulosi

Catalysts ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1011
Author(s):  
Paripok Phitsuwan ◽  
Sengthong Lee ◽  
Techly San ◽  
Khanok Ratanakhanokchai
Keyword(s):  
Glycoside Hydrolase ◽  
Cellulose Degradation ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Type I ◽  
Amorphous Cellulose ◽  
Carbohydrate Binding Modules ◽  
Glycoside Hydrolase Family 9 ◽  
Hydrolysis Of ◽  
Hydrolase Family

Glycoside hydrolase family 9 (GH9) endoglucanases are important enzymes for cellulose degradation. However, their activity on cellulose is diverse. Here, we cloned and expressed one GH9 enzyme (CalkGH9T) from Clostridium alkalicellulosi in Escherichia coli. CalkGH9T has a modular structure, containing one GH9 catalytic module, two family 3 carbohydrate binding modules, and one type I dockerin domain. CalkGH9T exhibited maximal activity at pH 7.0–8.0 and 55 °C and was resistant to urea and NaCl. It efficiently hydrolyzed carboxymethyl cellulose (CMC) but poorly degraded regenerated amorphous cellulose (RAC). Despite strongly binding to Avicel, CalkGH9T lacked the ability to hydrolyze this substrate. The hydrolysis of CMC by CalkGH9T produced a series of cello-oligomers, with cellotetraose being preferentially released. Similar proportions of soluble and insoluble reducing ends generated by hydrolysis of RAC indicated non-processive activity. Our study extends our knowledge of the molecular mechanism of cellulose hydrolysis by GH9 family endoglucanases with industrial relevance.

scholarly journals Molecular cloning and characterization of a novel β-1,3-xylanase possessing two putative carbohydrate-binding modules from a marine bacterium Vibrio sp. strain AX-4

2005 ◽  
Vol 388 (3) ◽  
pp. 949-957 ◽  
Author(s):  
Masashi KIYOHARA ◽  
Keishi SAKAGUCHI ◽  
Kuniko YAMAGUCHI ◽  
Toshiyoshi ARAKI ◽  
Takashi NAKAMURA ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Marine Bacterium ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Amino Acid Residues ◽  
Reading Frame ◽  
Carbohydrate Binding Modules ◽  
Hydrolysis Of ◽  
Hydrolase Family

We cloned a novel β-1,3-xylanase gene, consisting of a 1728-bp open reading frame encoding 576 amino acid residues, from a marine bacterium, Vibrio sp. strain AX-4. Sequence analysis revealed that the β-1,3-xylanase is a modular enzyme composed of a putative catalytic module belonging to glycoside hydrolase family 26 and two putative carbohydrate-binding modules belonging to family 31. The recombinant enzyme hydrolysed β-1,3-xylan to yield xylo-oligosaccharides with different numbers of xylose units, mainly xylobiose, xylotriose and xylotetraose. However, the enzyme did not hydrolyse β-1,4-xylan, β-1,4-mannan, β-1,4-glucan, β-1,3-xylobiose or p-nitrophenyl-β-xyloside. When β-1,3-xylo-oligosaccharides were used as the substrate, the kcat value of the enzyme for xylopentaose was found to be 40 times higher than that for xylotetraose, and xylotriose was extremely resistant to hydrolysis by the enzyme. A PSI-BLAST search revealed two possible catalytic Glu residues (Glu-138 as an acid/base catalyst and Glu-234 as a nucleophile), both of which are generally conserved in glycoside hydrolase superfamily A. Replacement of these two conserved Glu residues with Asp and Gln resulted in a significant decrease and complete loss of enzyme activity respectively, without a change in their CD spectra, suggesting that these Glu residues are the catalytic residues of β-1,3-xylanase. The present study also clearly shows that the non-catalytic putative carbohydrate-binding modules play an important role in the hydrolysis of insoluble β-1,3-xylan, but not that of soluble glycol-β-1,3-xylan. Furthermore, repeating a putative carbohydrate-binding module strongly enhanced the hydrolysis of the insoluble substrate.

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

scholarly journals The modular architecture of Cellvibrio japonicus mannanases in glycoside hydrolase families 5 and 26 points to differences in their role in mannan degradation

2003 ◽  
Vol 371 (3) ◽  
pp. 1027-1043 ◽  
Author(s):  
Deborah HOGG ◽  
Gavin PELL ◽  
Paul DUPREE ◽  
Florence GOUBET ◽  
Susana M. MARTÍN-ORÚE ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Plant Cell Wall ◽  
Catalytic Domain ◽  
Glycoside Hydrolases ◽  
Crystalline Cellulose ◽  
Carbohydrate Binding ◽  
Molecular Architecture ◽  
Catalytic Domains ◽  
Carbohydrate Binding Modules ◽  
Cellvibrio Japonicus

β-1,4-Mannanases (mannanases), which hydrolyse mannans and glucomannans, are located in glycoside hydrolase families (GHs) 5 and 26. To investigate whether there are fundamental differences in the molecular architecture and biochemical properties of GH5 and GH26 mannanases, four genes encoding these enzymes were isolated from Cellvibrio japonicus and the encoded glycoside hydrolases were characterized. The four genes, man5A, man5B, man5C and man26B, encode the mannanases Man5A, Man5B, Man5C and Man26B, respectively. Man26B consists of an N-terminal signal peptide linked via an extended serine-rich region to a GH26 catalytic domain. Man5A, Man5B and Man5C contain GH5 catalytic domains and non-catalytic carbohydrate-binding modules (CBMs) belonging to families 2a, 5 and 10; Man5C in addition contains a module defined as X4 of unknown function. The family 10 and 2a CBMs bound to crystalline cellulose and ivory nut crystalline mannan, displaying very similar properties to the corresponding family 10 and 2a CBMs from Cellvibrio cellulases and xylanases. CBM5 bound weakly to these crystalline polysaccharides. The catalytic domains of Man5A, Man5B and Man26B hydrolysed galactomannan and glucomannan, but displayed no activity against crystalline mannan or cellulosic substrates. Although Man5C was less active against glucomannan and galactomannan than the other mannanases, it did attack crystalline ivory nut mannan. All the enzymes exhibited classic endo-activity producing a mixture of oligosaccharides during the initial phase of the reaction, although their mode of action against manno-oligosaccharides and glucomannan indicated differences in the topology of the respective substrate-binding sites. This report points to a different role for GH5 and GH26 mannanases from C. japonicus. We propose that as the GH5 enzymes contain CBMs that bind crystalline polysaccharides, these enzymes are likely to target mannans that are integral to the plant cell wall, while GH26 mannanases, which lack CBMs and rapidly release mannose from polysaccharides and oligosaccharides, target the storage polysaccharide galactomannan and manno-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.

Glycoside hydrolase carbohydrate-binding modules as molecular probes for the analysis of plant cell wall polymers

2004 ◽  
Vol 326 (1) ◽  
pp. 49-54 ◽  
Author(s):  
Lesley McCartney ◽  
Harry J Gilbert ◽  
David N Bolam ◽  
Alisdair B Boraston ◽  
J.Paul Knox
Keyword(s):  
Cell Wall ◽  
Plant Cell ◽  
Glycoside Hydrolase ◽  
Plant Cell Wall ◽  
Molecular Probes ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Modules ◽  
Cell Wall Polymers
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