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

McKee, Lauren S.; Peña, María J.; Rogowski, Artur; Jackson, Adam; Lewis, Richard J.; York, William S.; Krogh, Kristian B. R. M.; Viksø-Nielsen, Anders; Skjøt, Michael; Gilbert, Harry J.; Marles‐Wright, Jon

doi:10.1073/pnas.1117686109

Cited by 74 publications

(61 citation statements)

References 19 publications

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“…Of particular note is subfamily 36, which has demonstrated activity against disubstituted (1,2-and 1,3-arabinofuranoside) xylopyranose residues (26,27). These residues are typically recalcitrant to enzymatic attack, and as such, this subfamily has significant biotechnological interest.…”

Section: Resultsmentioning

confidence: 99%

Dividing the Large Glycoside Hydrolase Family 43 into Subfamilies: a Motivation for Detailed Enzyme Characterization

Mewis

Lenfant

Lombard

et al. 2016

Appl Environ Microbiol

195

167

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dThe rapid rise in DNA sequencing has led to an expansion in the number of glycoside hydrolase (GH) families. The GH43 family currently contains ␣-L-arabinofuranosidase, ␤-D-xylosidase, ␣-L-arabinanase, and ␤-D-galactosidase enzymes for the debranching and degradation of hemicellulose and pectin polymers. Many studies have revealed finer details about members of GH43 that necessitate the division of GH43 into subfamilies, as was done previously for the GH5 and GH13 families. The work presented here is a robust subfamily classification that assigns over 91% of all complete GH43 domains into 37 subfamilies that correlate with conserved sequence residues and results of biochemical assays and structural studies. Furthermore, cooccurrence analysis of these subfamilies and other functional modules revealed strong associations between some GH43 subfamilies and CBM6 and CBM13 domains. Cooccurrence analysis also revealed the presence of proteins containing up to three GH43 domains and belonging to different subfamilies, suggesting significant functional differences for each subfamily. Overall, the subfamily analysis suggests that the GH43 enzymes probably display a hitherto underestimated variety of subtle specificity features that are not apparent when the enzymes are assayed with simple synthetic substrates, such as pNP-glycosides. Carbohydrates serve a range of functional purposes in biological systems, including energy storage, signal transduction, and intracellular trafficking, among others (1). Importantly, carbohydrates are the main end product of plant primary production, representing a large of majority of carbon fixation by plants (2). As a photosynthetically renewable form of fixed carbon, plant biomass represents a prime target for the replacement of petroleumderived fuels for future sustainability efforts. The enzymatic degradation and modification of carbohydrates have thus been cast to the forefront of biofuel production research (3).As functional efforts to discover plant cell wall polysaccharide (PCWP)-degrading enzymes identify novel activities and mechanisms (4, 5), it is important to derive and maintain a concise classification system for these enzymes. A sequence-based classification of carbohydrate-active enzymes (CAZymes) began in 1991 (6), with the classification of 35 families of glycoside hydrolases (GHs). Today the CAZy database (7) comprises 5 separate enzyme classes, namely, the aforementioned GHs, glycosyltransferases (GTs), polysaccharide lyases (PLs), carbohydrate esterases (CEs), and auxiliary activities (AAs), as well as associated carbohydrate binding modules (CBMs), that together correspond to over 530,000 individual sequences (at the time of submission of this article). The largest of these classes are the glycoside hydrolases, currently represented by over 241,000 sequences classified into 133 families based on amino acid sequence similarity.The rapid advancements in DNA sequencing over the past decade have exponentially increased the number of sequences assigned to each family. Henc...

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Section: Resultsmentioning

confidence: 99%

Dividing the Large Glycoside Hydrolase Family 43 into Subfamilies: a Motivation for Detailed Enzyme Characterization

Mewis

Lenfant

Lombard

et al. 2016

Appl Environ Microbiol

195

167

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“…xylanase activity was introduced into a GH43 arabinofuranosidase (38), and endo-mannanase activity was introduced into a GH26 exo-acting enzyme (39). Further careful biophysical characterization of other members of the GH5_26 subfamily is now required to assess whether the quaternary structure controls the substrate specificity of the entire GH5_26 subfamily.…”

Section: Discussionmentioning

confidence: 99%

The Quaternary Structure of a Glycoside Hydrolase Dictates Specificity toward β-Glucans

Lafond

Sulzenbacher

Freyd

et al. 2016

Journal of Biological Chemistry

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In the Carbohydrate-Active Enzyme (CAZy) database, glycoside hydrolase family 5 (GH5) is a large family with more than 6,000 sequences. Among the 51 described GH5 subfamilies, subfamily GH5_26 contains members that display either endo-␤(1,4)-glucanase or ␤(1,3;1,4)-glucanase activities. In this study, we focused on the GH5_26 enzyme from Saccharophagus degradans (SdGluc5_26A), a marine bacterium known for its capacity to degrade a wide diversity of complex polysaccharides. SdGluc5_26A displays lichenase activity toward ␤(1,3; 1,4)-glucans with a side cellobiohydrolase activity toward ␤(1, 4)-glucans. The three-dimensional structure of SdGluc5_26A adopts a stable trimeric quaternary structure also observable in solution. The N-terminal region of SdGluc5_26A protrudes into the active site of an adjacent monomer. To understand whether this occupation of the active site could influence its activity, we conducted a comprehensive enzymatic characterization of SdGluc5_26A and of a mutant truncated at the N terminus. Ligand complex structures and kinetic analyses reveal that the N terminus governs the substrate specificity of SdGluc5_26A. Its deletion opens the enzyme cleft at the ؊3 subsite and turns the enzyme into an endo-␤(1,4)-glucanase. This study demonstrates that experimental approaches can reveal structure-function relationships out of reach of current bioinformatic predictions.

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“…ABFs that release Araf from doubly substituted Xylp are specifically releasing either 1,2-or 1,3-linked given the surfix d2 or d3, respectively (Cartmell et al, 2011;McKee et al, 2012;Pouvreau et al, 2011;Van Laere et al, 1997.…”

Section: Classification Of -L-arabinofuranosidasesmentioning

confidence: 99%

GH62 arabinofuranosidases: Structure, function and applications

Wilkens

Andersen

Dumon

et al. 2017

Biotechnology Advances

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Motivated by industrial demands and ongoing scientific discoveries continuous efforts are made to identify and create improved biocatalysts dedicated to plant biomass conversion. --1,3 arabinofuranosyl specific -L-arabinofuranosidases (EC 3.2.1.55) are debranching enzymes catalyzing hydrolytic release -L-arabinofuranosyl residues, which decorate xylan or arabinan backbones in lignocellulosic and pectin constituents of plant cell walls. The CAZy database classifies -L-arabinofuranosidases in Glycoside Hydrolase (GH) families GH2, GH3, GH43, GH51, GH54 and GH62. Only GH62 contains exclusively -L-arabinofuranosidases and these are of fungal and bacterial origin. Twenty-two GH62 enzymes out of 223 entries in the CAZy database have been characterized and very recently new knowledge was acquired with regard to crystal structures, substrate specificities, and phylogenetics, which overall provides novel insights into structure/function relationships of GH62. Overall GH62 -L-arabinofuranosidases are believed to play important roles in nature by acting in synergy with several cell wall degrading enzymes and members of GH62 represent promising candidates for biotechnological improvements of biofuel production and in various biorefinery applications.3

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

Cited by 74 publications

References 19 publications

Dividing the Large Glycoside Hydrolase Family 43 into Subfamilies: a Motivation for Detailed Enzyme Characterization

Dividing the Large Glycoside Hydrolase Family 43 into Subfamilies: a Motivation for Detailed Enzyme Characterization

The Quaternary Structure of a Glycoside Hydrolase Dictates Specificity toward β-Glucans

GH62 arabinofuranosidases: Structure, function and applications

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