Structural enzymology reveals the molecular basis of substrate regiospecificity and processivity of an exemplar bacterial glycoside hydrolase family 74 endo-xyloglucanase

Paenibacillus odorifer produces a single multimodular enzyme containing a glycoside hydrolase (GH) family 74 module (AIQ73809). Recombinant production and characterization of the GH74 module (PoGH74cat) revealed a highly specific, processive endo-xyloglucanase that can hydrolyze the polysaccharide backbone at both branched and unbranched positions. X-ray crystal structures obtained for the free enzyme and oligosaccharide complexes evidenced an extensive hydrophobic binding platform — the first in GH74 extending from subsites −4 to +6 — and unique mobile active-site loops. Site-directed mutagenesis revealed that glycine-476 was uniquely responsible for the promiscuous backbone-cleaving activity of PoGH74cat; replacement with tyrosine, which is conserved in many GH74 members, resulted in exclusive hydrolysis at unbranched glucose units. Likewise, systematic replacement of the hydrophobic platform residues constituting the positive subsites indicated their relative contributions to the processive mode of action. Specifically, W347 (+3 subsite) and W348 (+5 subsite) are essential for processivity, while W406 (+2 subsite) and Y372 (+6 subsite) are not strictly essential, but aid processivity.

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Heterologous expression, crystallization and preliminary X-ray characterization of CcCel6C, a glycoside hydrolase family 6 enzyme from the basidiomyceteCoprinopsis cinerea

Acta Crystallographica Section F Structural Biology and Crystallization Communications ◽

10.1107/s174430910804284x ◽

2009 ◽

Vol 65 (2) ◽

pp. 140-143 ◽

Cited By ~ 5

Author(s):

Yuma Kurakata ◽

Takashi Tonozuka ◽

Yuan Liu ◽

Satoshi Kaneko ◽

Atsushi Nishikawa ◽

...

Keyword(s):

Heterologous Expression ◽

Glycoside Hydrolase ◽

Glycoside Hydrolase Family ◽

X Ray ◽

Hydrolase Family

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Characterization of Fusarium oxysporum β-1,6-Galactanase, an Enzyme That Hydrolyzes Larch Wood Arabinogalactan

Applied and Environmental Microbiology ◽

10.1128/aem.02101-06 ◽

2007 ◽

Vol 73 (9) ◽

pp. 3109-3112 ◽

Cited By ~ 17

Author(s):

Tatsuji Sakamoto ◽

Yuya Taniguchi ◽

Shiho Suzuki ◽

Hideshi Ihara ◽

Haruhiko Kawasaki

Keyword(s):

Fusarium Oxysporum ◽

Glycoside Hydrolase ◽

Recombinant Enzyme ◽

Glycoside Hydrolase Family ◽

Type Ii ◽

High Similarity ◽

Degrading Enzyme ◽

Larch Wood ◽

Hydrolase Family

ABSTRACT A type II arabinogalactan-degrading enzyme (FoGal1) was purified from Fusarium oxysporum 12S, and the corresponding cDNA was isolated. FoGal1 had high similarity to enzymes of glycoside hydrolase family 5. Treatment of larch wood arabinogalactan with the recombinant enzyme indicated that FoGal1 is a β-1,6-galactanase that preferentially debranches β-1,6-galactobiose from the substrate.

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Crystallization and preliminary X-ray diffraction study of two complexes of a TAXI-type xylanase inhibitor with glycoside hydrolase family 11 xylanases fromAspergillus nigerandBacillus subtilis

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444903029330 ◽

2004 ◽

Vol 60 (3) ◽

pp. 555-557 ◽

Cited By ~ 8

Author(s):

Stefaan Sansen ◽

Camiel J. De Ranter ◽

Kurt Gebruers ◽

Kristof Brijs ◽

Christophe M. Courtin ◽

...

Keyword(s):

Diffraction Study ◽

Glycoside Hydrolase ◽

Glycoside Hydrolase Family ◽

X Ray Diffraction ◽

X Ray ◽

Xylanase Inhibitor ◽

Hydrolase Family

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Characterization of the endoglucanase and glucomannanase activities of a glycoside hydrolase family 45 protein from Penicillium decumbens 114-2

The Journal of General and Applied Microbiology ◽

10.2323/jgam.56.223 ◽

2010 ◽

Vol 56 (3) ◽

pp. 223-229 ◽

Cited By ~ 24

Author(s):

Guodong Liu ◽

Xiaomin Wei ◽

Yuqi Qin ◽

Yinbo Qu

Keyword(s):

Glycoside Hydrolase ◽

Glycoside Hydrolase Family ◽

Penicillium Decumbens ◽

Hydrolase Family

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

10.1101/2021.10.27.466152 ◽

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.

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