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

Vandermarliere, Elien; Bourgois, Tine M.; Winn, Martyn; Campenhout, Steven Van; Volckaert, Guido; Delcour, Jan A.; Strelkov, S.V.; Rabijns, A.; Courtin, Christophe M.

doi:10.1042/bj20081256

Cited by 78 publications

(69 citation statements)

References 38 publications

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“…Gilbert, unpublished data) reveals an extended substrate-binding pocket that interacts with both O2-and O3-linked Araf. By contrast, an arabinoxylan-specific GH43 arabinofuranosidase, which removes O2-or O3-linked Araf side chains from singularly substituted Xyl residues, contains a small substrate-binding pocket embedded in a shallow cleft that is optimized to bind the 3-fold helical structure of the xylan backbone (Vandermarliere et al, 2009). Recent protein crystallographic studies have shown that xylan side chains can be accommodated and can actually be exploited as specificity determinants (Pell et al, 2004;Vardakou et al, 2005), while a GH5 xylanase displays an absolute requirement for 4-O-methyl-D-GlcUA appended to the Xyl positioned at the 22 subsite (Vrsanska et al, 2007).…”

Section: Xylan Degradationmentioning

confidence: 99%

The Biochemistry and Structural Biology of Plant Cell Wall Deconstruction

2010

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Section: Xylan Degradationmentioning

confidence: 99%

The Biochemistry and Structural Biology of Plant Cell Wall Deconstruction

2010

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“…CjAbf43A displays a five-bladed ␤-propeller fold (Fig. 4), typical of GH43 enzymes (8,10,11,32,38). It has a cylindrical shape with a diameter and height of 35 Å.…”

Section: Three-dimensional Structure Of Cjabf43amentioning

confidence: 99%

The Structure and Function of an Arabinan-specific α-1,2-Arabinofuranosidase Identified from Screening the Activities of Bacterial GH43 Glycoside Hydrolases

Cartmell

McKee

Peña

et al. 2011

Journal of Biological Chemistry

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Reflecting the diverse chemistry of plant cell walls, microorganisms that degrade these composite structures synthesize an array of glycoside hydrolases. These enzymes are organized into sequence-, mechanism-, and structure-based families. Genomic data have shown that several organisms that degrade the plant cell wall contain a large number of genes encoding family 43 (GH43) glycoside hydrolases. Here we report the biochemical properties of the GH43 enzymes of a saprophytic soil bacterium, Cellvibrio japonicus, and a human colonic symbiont, Bacteroides thetaiotaomicron. The data show that C. japonicus uses predominantly exo-acting enzymes to degrade arabinan into arabinose, whereas B. thetaiotaomicron deploys a combination of endo-and side chain-cleaving glycoside hydrolases. Both organisms, however, utilize an arabinan-specific ␣-1,2-arabinofuranosidase in the degradative process, an activity that has not previously been reported. The enzyme can cleave ␣-1,2-arabinofuranose decorations in single or double substitutions, the latter being recalcitrant to the action of other arabinofuranosidases. The crystal structure of the C. japonicus arabinan-specific ␣-1,2-arabinofuranosidase, CjAbf43A, displays a fivebladed ␤-propeller fold. The specificity of the enzyme for arabinan is conferred by a surface cleft that is complementary to the helical backbone of the polysaccharide. The specificity of CjAbf43A for ␣-1,2-L-arabinofuranose side chains is conferred by a polar residue that orientates the arabinan backbone such that O2 arabinose decorations are directed into the active site pocket. A shelflike structure adjacent to the active site pocket accommodates O3 arabinose side chains, explaining how the enzyme can target O2 linkages that are components of single or double substitutions.

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“…Endo and backbone exo-acting enzymes are closely related; in nature, short loop extensions have led to the conversion of endo-glycanases into exo-glycanases that target the polysaccharide backbone (6)(7)(8). The active site of enzymes that cleave sugars decorating the polysaccharide backbone displays a pocket topology in which the target side chain is positioned (9,10). In contrast to enzymes that target the backbone of complex carbohydrates, the introduction of endo activity into a glycoside hydrolase that removes polysaccharide side chains has not been reported.…”

Section: For Review)mentioning

confidence: 99%

Introducing endo-xylanase activity into an exo-acting arabinofuranosidase that targets side chains

McKee

Peña

Rogowski

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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The degradation of the plant cell wall by glycoside hydrolases is central to environmentally sustainable industries. The major polysaccharides of the plant cell wall are cellulose and xylan, a highly decorated β-1,4-xylopyranose polymer. Glycoside hydrolases displaying multiple catalytic functions may simplify the enzymes required to degrade plant cell walls, increasing the industrial potential of these composite structures. Here we test the hypothesis that glycoside hydrolase family 43 (GH43) provides a suitable scaffold for introducing additional catalytic functions into enzymes that target complex structures in the plant cell wall. We report the crystal structure of Humicola insolens AXHd3 (HiAXHd3), a GH43 arabinofuranosidase that hydrolyses O3-linked arabinose of doubly substituted xylans, a feature of the polysaccharide that is recalcitrant to degradation. HiAXHd3 displays an N-terminal five-bladed β-propeller domain and a C-terminal β-sandwich domain. The interface between the domains comprises a xylan binding cleft that houses the active site pocket. Substrate specificity is conferred by a shallow arabinose binding pocket adjacent to the deep active site pocket, and through the orientation of the xylan backbone. Modification of the rim of the active site introduces endo-xylanase activity, whereas the resultant enzyme variant, Y166A, retains arabinofuranosidase activity. These data show that the active site of HiAXHd3 is tuned to hydrolyse arabinofuranosyl or xylosyl linkages, and it is the topology of the distal regions of the substrate binding surface that confers specificity. This report demonstrates that GH43 provides a platform for generating bespoke multifunctional enzymes that target industrially significant complex substrates, exemplified by the plant cell wall. biofuels | biotechnology | protein engineering | structural biology

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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

Cited by 78 publications

References 38 publications

The Biochemistry and Structural Biology of Plant Cell Wall Deconstruction

The Biochemistry and Structural Biology of Plant Cell Wall Deconstruction

The Structure and Function of an Arabinan-specific α-1,2-Arabinofuranosidase Identified from Screening the Activities of Bacterial GH43 Glycoside Hydrolases

Introducing endo-xylanase activity into an exo-acting arabinofuranosidase that targets side chains

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