Structural and biochemical characterization of novel bacterial α-galactosidases belonging to glycoside hydrolase family 31

2015 ◽  
Vol 469 (1) ◽  
pp. 145-158 ◽  
Author(s):  
Takatsugu Miyazaki ◽  
Yuichi Ishizaki ◽  
Megumi Ichikawa ◽  
Atsushi Nishikawa ◽  
Takashi Tonozuka
Keyword(s):  
Substrate Specificity ◽  
Crystal Structures ◽  
Glycoside Hydrolase ◽  
Biochemical Characterization ◽  
Glycoside Hydrolase Family ◽  
Galactosidase Activity ◽  
Gh31 Family ◽  
Glycoside Hydrolase Family 31 ◽  
Hydrolase Family

We identified two bacterial enzymes as the first members that displayed α-galactosidase activity and the crystal structures provided insights into their novel substrate specificity. This is the first report of α-galactosidases which belong to the GH31 family.

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.

Characterization of a Glycoside Hydrolase Family 31 α-Glucosidase Involved in Starch Utilization inPodospora anserina

2013 ◽  
Vol 77 (10) ◽  
pp. 2117-2124 ◽  
Author(s):  
Kyung-Mo SONG ◽  
Masayuki OKUYAMA ◽  
Kazuyuki KOBAYASHI ◽  
Haruhide MORI ◽  
Atsuo KIMURA
Keyword(s):  
Glycoside Hydrolase ◽  
Glycoside Hydrolase Family ◽  
Starch Utilization ◽  
Glycoside Hydrolase Family 31 ◽  
Hydrolase Family

scholarly journals Structural and functional studies of the glycoside hydrolase family 3 β-glucosidase Cel3A from the moderately thermophilic fungusRasamsonia emersonii

2016 ◽  
Vol 72 (7) ◽  
pp. 860-870 ◽  
Author(s):  
Mikael Gudmundsson ◽  
Henrik Hansson ◽  
Saeid Karkehabadi ◽  
Anna Larsson ◽  
Ingeborg Stals ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Biochemical Characterization ◽  
Cellulosic Biomass ◽  
Glycoside Hydrolase Family ◽  
Hypocrea Jecorina ◽  
Moderately Thermophilic ◽  
Functional Studies ◽  
Excellent Candidate ◽  
Hydrolase Family

The filamentous fungusHypocrea jecorinaproduces a number of cellulases and hemicellulases that act in a concerted fashion on biomass and degrade it into monomeric or oligomeric sugars. β-Glucosidases are involved in the last step of the degradation of cellulosic biomass and hydrolyse the β-glycosidic linkage between two adjacent molecules in dimers and oligomers of glucose. In this study, it is shown that substituting the β-glucosidase fromH. jecorina(HjCel3A) with the β-glucosidase Cel3A from the thermophilic fungusRasamsonia emersonii(ReCel3A) in enzyme mixtures results in increased efficiency in the saccharification of lignocellulosic materials. Biochemical characterization ofReCel3A, heterologously produced inH. jecorina, reveals a preference for disaccharide substrates over longer gluco-oligosaccharides. Crystallographic studies ofReCel3A revealed a highly N-glycosylated three-domain dimeric protein, as has been observed previously for glycoside hydrolase family 3 β-glucosidases. The increased thermal stability and saccharification yield and the superior biochemical characteristics ofReCel3A compared withHjCel3A and mixtures containingHjCel3A makeReCel3A an excellent candidate for addition to enzyme mixtures designed to operate at higher temperatures.

scholarly journals Cloning and biochemical characterization of the fucanase FcnA: definition of a novel glycoside hydrolase family specific for sulfated fucans

Glycobiology ◽  
2006 ◽  
Vol 16 (11) ◽  
pp. 1021-1032 ◽  
Author(s):  
Sébastien Colin ◽  
Estelle Deniaud ◽  
Murielle Jam ◽  
Valérie Descamps ◽  
Yann Chevolot ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Biochemical Characterization ◽  
Glycoside Hydrolase Family ◽  
Definition Of ◽  
Hydrolase Family

Biochemical characterization of a glycoside hydrolase family 61 endoglucanase from Aspergillus kawachii

2008 ◽  
Vol 77 (6) ◽  
pp. 1279-1285 ◽  
Author(s):  
Takuya Koseki ◽  
Yuichiro Mese ◽  
Shinya Fushinobu ◽  
Kazuo Masaki ◽  
Tsutomu Fujii ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Biochemical Characterization ◽  
Glycoside Hydrolase Family ◽  
Aspergillus Kawachii ◽  
Hydrolase Family

scholarly journals A novel member of glycoside hydrolase family 30 subfamily 8 with altered substrate specificity

2014 ◽  
Vol 70 (11) ◽  
pp. 2950-2958 ◽  
Author(s):  
Franz J. St John ◽  
Diane Dietrich ◽  
Casey Crooks ◽  
Edwin Pozharski ◽  
Javier M. González ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Glycoside Hydrolase ◽  
Biochemical Characterization ◽  
Amino Acid Identity ◽  
Glycoside Hydrolase Family ◽  
High Sequence Identity ◽  
Loop Region ◽  
Hydrolysis Of ◽  
Hydrolase Family ◽  
Glycoside Hydrolase Family 30

Endoxylanases classified into glycoside hydrolase family 30 subfamily 8 (GH30-8) are known to hydrolyze the hemicellulosic polysaccharide glucuronoxylan (GX) but not arabinoxylan or neutral xylooligosaccharides. This is owing to the specificity of these enzymes for the α-1,2-linked glucuronate (GA) appendage of GX. Limit hydrolysis of this substrate produces a series of aldouronates each containing a single GA substituted on the xylose penultimate to the reducing terminus. In this work, the structural and biochemical characterization of xylanase 30A fromClostridium papyrosolvens(CpXyn30A) is presented. This xylanase possesses a high degree of amino-acid identity to the canonical GH30-8 enzymes, but lacks the hallmark β8–α8 loop region which in part defines the function of this GH30 subfamily and its role in GA recognition.CpXyn30A is shown to have a similarly low activity on all xylan substrates, while hydrolysis of xylohexaose revealed a competing transglycosylation reaction. These findings are directly compared with the model GH30-8 enzyme fromBacillus subtilis, XynC. Despite its high sequence identity to the GH30-8 enzymes,CpXyn30A does not have any apparent specificity for the GA appendage. These findings confirm that the typically conserved β8–α8 loop region of these enzymes influences xylan substrate specificity but not necessarily β-1,4-xylanase function.

scholarly journals Structural and Biochemical Characterization of Glycoside Hydrolase Family 79 β-Glucuronidase fromAcidobacterium capsulatum

2012 ◽  
Vol 287 (17) ◽  
pp. 14069-14077 ◽  
Author(s):  
Mari Michikawa ◽  
Hitomi Ichinose ◽  
Mitsuru Momma ◽  
Peter Biely ◽  
Seino Jongkees ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Biochemical Characterization ◽  
Glycoside Hydrolase Family ◽  
Hydrolase Family

scholarly journals Structural and enzymatic characterization of a glycoside hydrolase family 31 α-xylosidase from Cellvibrio japonicus involved in xyloglucan saccharification

2011 ◽  
Vol 436 (3) ◽  
pp. 567-580 ◽  
Author(s):  
Johan Larsbrink ◽  
Atsushi Izumi ◽  
Farid M. Ibatullin ◽  
Azadeh Nakhai ◽  
Harry J. Gilbert ◽  
...  
Keyword(s):  
Cell Wall ◽  
Glycoside Hydrolase ◽  
Plant Cell Wall ◽  
Biomass Conversion ◽  
Primary Cell Wall ◽  
Glycoside Hydrolase Family ◽  
Cellvibrio Japonicus ◽  
Glycoside Hydrolase Family 31 ◽  
Hydrolase Family

The desire for improved methods of biomass conversion into fuels and feedstocks has re-awakened interest in the enzymology of plant cell wall degradation. The complex polysaccharide xyloglucan is abundant in plant matter, where it may account for up to 20% of the total primary cell wall carbohydrates. Despite this, few studies have focused on xyloglucan saccharification, which requires a consortium of enzymes including endo-xyloglucanases, α-xylosidases, β-galactosidases and α-L-fucosidases, among others. In the present paper, we show the characterization of Xyl31A, a key α-xylosidase in xyloglucan utilization by the model Gram-negative soil saprophyte Cellvibrio japonicus. CjXyl31A exhibits high regiospecificity for the hydrolysis of XGOs (xylogluco-oligosaccharides), with a particular preference for longer substrates. Crystallographic structures of both the apo enzyme and the trapped covalent 5-fluoro-β-xylosyl-enzyme intermediate, together with docking studies with the XXXG heptasaccharide, revealed, for the first time in GH31 (glycoside hydrolase family 31), the importance of a PA14 domain insert in the recognition of longer oligosaccharides by extension of the active-site pocket. The observation that CjXyl31A was localized to the outer membrane provided support for a biological model of xyloglucan utilization by C. japonicus, in which XGOs generated by the action of a secreted endo-xyloglucanase are ultimately degraded in close proximity to the cell surface. Moreover, the present study diversifies the toolbox of glycosidases for the specific modification and saccharification of cell wall polymers for biotechnological applications.

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