The Structure of an Inverting GH43 β-Xylosidase from Geobacillus stearothermophilus with its Substrate Reveals the Role of the Three Catalytic Residues

Brüx, Christian; Ben-David, Alon; Shallom-Shezifi, D.; Leon, Maya; Niefind, Karsten; Shoham, G.; Shoham, Yuval; Schomburg, Dietmar

doi:10.1016/j.jmb.2006.03.005

Cited by 134 publications

(157 citation statements)

References 39 publications

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“…The engineered enzyme is thus able to make productive interactions with L-furanose and D-pyranose pentose sugars that display different stereochemistry at C4. This has some resonance with studies showing that GH3 and GH43 β-xylosidases can display low levels of arabinofuranosidase activity (12,15), whereas a GH51 arabinofuranosidase is also able to hydrolyze β-xylosyl linkages (18). The capacity to hydrolyze both α-L-Araf and β-D-Xylp linkages is consistent with the observation that the two sugars share considerable spatial similarity, which is particularly evident when one considers the free rotation of the Araf exocyclic C5 hydroxymethyl group.…”

Section: Discussionsupporting

confidence: 85%

“…A distinctive feature of GH43 is that the active site of the β-xylosidases and arabinofuranosidases are highly conserved (10) (Fig. S1), consistent with the observation that β-xylosidases and some arabinofuranosidases are capable of also cleaving α-arabinofuranosyl and β-xylopyranosyl linkages, respectively (12,15) . It would seem, therefore, that substrate binding regions distal to the active site contribute to the specifi-city of GH43 enzymes.…”

Section: For Review)supporting

confidence: 78%

“…The active site of HiAXHd3, located in the center of the xylotriose binding cleft, contains a constellation of carboxylate residues, Asp43, Asp167, and Glu216, which are invariant within GH43. The equivalent residues have been shown to comprise the catalytic amino acids in other members of the family (7,(11)(12)(13) (Fig. S3).…”

Section: Hiaxhd3mentioning

confidence: 99%

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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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Section: Discussionsupporting

confidence: 85%

Section: For Review)supporting

confidence: 78%

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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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“…japonicus-Biochemical analysis of the two exo-␣-1,5-arabinofuranosidases, CJA_3012 and CJA_0806 ( Table 2), showed that both enzymes displayed the highest catalytic activity against linear arabinan and arabinooligosaccharides, releasing exclusively arabinose, probably from the non-reducing end (all exo-acting GH43 enzymes attack the non-reducing end of polysaccharides (8,11,31,32)). The two enzymes released only very small amounts of arabinose from sugar beet, reflecting the limited concentration of terminal undecorated ␣-1,5-arabinofuranose units in this highly branched polysaccharide (data not shown).…”

Section: Characterization Of Arabinan-degrading Enzymesmentioning

confidence: 99%

“…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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2012

Fungi and Lignocellulosic Biomass

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The Structure of an Inverting GH43 β-Xylosidase from Geobacillus stearothermophilus with its Substrate Reveals the Role of the Three Catalytic Residues

Cited by 134 publications

References 39 publications

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

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

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

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