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

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.

A Novel Multifunctional Arabinofuranosidase/Endo-xylanase/β-Xylosidase GH43 from Paenibacillus curdlanolyticus B-6 and Its Synergistic Action to Produce Arabinose and Xylose from Cereal Arabinoxylan

The PcAxy43B is a modular protein comprising a catalytic domain of glycoside hydrolase family 43 (GH43), a family 6 carbohydrate-binding module (CBM6) and a family 36 carbohydrate-binding module (CBM36) and found to be a novel multifunctional xylanolytic enzyme from Paenibacillus curdlanolyticus B-6. This enzyme exhibited α-L-arabinofuranosidase, endo-xylanase and β-D-xylosidase activities. α-L-Arabinofuranosidase of PcAxy43B revealed the new property of GH43, which released arabinose from the short-chain arabinoxylo-oligosaccharide (AXOS) and cereal arabinoxylan, and from both sides of the xylose residues of AXOS, which usually obstruct the action of xylanolytic enzymes. The PcAxy43B liberated series of xylo-oligosaccharides (XOSs) from birchwood xylan and xylohexaose, indicating that PcAxy43B exhibited endo-xylanase activity. The PcAxy43B produced xylose from xylobiose and reacted with p -nitrophenyl-β-D-xylopyranoside as a result of β-xylosidase activity. The PcAxy43B effectively released arabinose together with XOSs and xylose from the highly arabinosyl-substituted rye arabinoxylan. Moreover, PcAxy43B showed significant synergistic action with a trifunctional endo-xylanase/β-xylosidase/α-L-arabinofuranosidase PcAxy43A and an endo-xylanase Xyn10C from the strain B-6, in which almost all products produced from rye arabinoxylan by these combined enzymes were arabinose and xylose. In addition, the presence of CBM36 was found to be necessary for the endo-xylanase property of PcAxy43B. The PcAxy43B is capable of hydrolysing untreated cereal biomass, corn hull and rice straw into XOSs and xylose. Hence, PcAxy43B, the significant accessory multifunctional xylanolytic enzyme, is a potential candidate for application in the saccharification of cereal biomass. IMPORTANCE Enzymatic saccharification of cereal biomass is a strategy for the production of fermented sugars from low-price raw materials. In the present study, PcAxy43B from P. curdlanolyticus B-6 was found to be a novel multifunctional α-L-arabinofuranosidase/endo-xylanase/β-D-xylosidase enzyme of the glycoside hydrolase family 43. It is effective in releasing arabinose, xylose and XOSs from the highly arabinosyl-substituted rye arabinoxylan, which is usually resistant to hydrolysis by xylanolytic enzymes. Moreover, almost all products produced from rye arabinoxylan by the combination of PcAxy43B with trifunctional xylanolytic enzyme PcAxy43A and endo-xylanase Xyn10C from the strain B-6 were arabinose and xylose, which can be used to produce several value-added products. In addition, PcAxy43B is capable of hydrolysing untreated cereal biomass into XOSs and xylose. Thus, PcAxy43B is an important multifunctional xylanolytic enzyme with high potential in biotechnology.

Two distinct catalytic pathways for GH43 xylanolytic enzymes unveiled by X-ray and QM/MM simulations

Xylanolytic enzymes from glycoside hydrolase family 43 (GH43) are involved in the breakdown of hemicellulose, the second most abundant carbohydrate in plants. Here, we kinetically and mechanistically describe the non-reducing-end xylose-releasing exo-oligoxylanase activity and report the crystal structure of a native GH43 Michaelis complex with its substrate prior to hydrolysis. Two distinct calcium-stabilized conformations of the active site xylosyl unit are found, suggesting two alternative catalytic routes. These results are confirmed by QM/MM simulations that unveil the complete hydrolysis mechanism and identify two possible reaction pathways, involving different transition state conformations for the cleavage of xylooligosaccharides. Such catalytic conformational promiscuity in glycosidases is related to the open architecture of the active site and thus might be extended to other exo-acting enzymes. These findings expand the current general model of catalytic mechanism of glycosidases, a main reaction in nature, and impact on our understanding about their interaction with substrates and inhibitors.

Identification of a Key Enzyme for the Hydrolysis of β-(1→3)-Xylosyl Linkage in Red Alga Dulse Xylooligosaccharide from Bifidobacterium Adolescentis

Red alga dulse possesses a unique xylan, which is composed of a linear β-(1→3)/β-(1→4)-xylosyl linkage. We previously prepared characteristic xylooligosaccharide (DX3, (β-(1→3)-xylosyl-xylobiose)) from dulse. In this study, we evaluated the prebiotic effect of DX3 on enteric bacterium. Although DX3 was utilized by Bacteroides sp. and Bifidobacterium adolescentis, Bacteroides Ksp. grew slowly as compared with β-(1→4)-xylotriose (X3) but B. adolescentis grew similar to X3. Therefore, we aimed to find the key DX3 hydrolysis enzymes in B. adolescentis. From bioinformatics analysis, two enzymes from the glycoside hydrolase family 43 (BAD0423: subfamily 12 and BAD0428: subfamily 11) were selected and expressed in Escherichia coli. BAD0423 hydrolyzed β-(1→3)-xylosyl linkage in DX3 with the specific activity of 2988 mU/mg producing xylose (X1) and xylobiose (X2), and showed low activity on X2 and X3. BAD0428 showed high activity on X2 and X3 producing X1, and the activity of BAD0428 on DX3 was 1298 mU/mg producing X1. Cooperative hydrolysis of DX3 was found in the combination of BAD0423 and BAD0428 producing X1 as the main product. From enzymatic character, hydrolysis of X3 was completed by one enzyme BAD0428, whereas hydrolysis of DX3 needed more than two enzymes.

Two Novel α-L-Arabinofuranosidases from Bifidobacterium longum subsp. longum Belonging to Glycoside Hydrolase Family 43 Cooperatively Degrade Arabinan

ABSTRACT Arabinose-containing poly- or oligosaccharides are suitable carbohydrate sources for Bifidobacterium longum subsp. longum. However, their degradation pathways are poorly understood. In this study, we cloned and characterized the previously uncharacterized glycoside hydrolase family 43 (GH43) enzymes B. longum subsp. longum ArafC (BlArafC; encoded by BLLJ_1852) and B. longum subsp. longum ArafB (BlArafB; encoded by BLLJ_1853) from B. longum subsp. longum JCM 1217. Both enzymes exhibited α-l-arabinofuranosidase activity toward p-nitrophenyl-α-l-arabinofuranoside but no activity toward p-nitrophenyl-β-d-xylopyranoside. The specificities of the two enzymes for l-arabinofuranosyl linkages were different. BlArafC catalyzed the hydrolysis of α1,2- and α1,3-l-arabinofuranosyl linkages found on the side chains of both arabinan and arabinoxylan. It released l-arabinose 100 times faster from arabinan than from arabinoxylan but did not act on arabinogalactan. On the other hand, BlArafB catalyzed the hydrolysis of the α1,5-l-arabinofuranosyl linkage found on the arabinan backbone. It released l-arabinose from arabinan but not from arabinoxylan or arabinogalactan. Coincubation of BlArafC and BlArafB revealed that these two enzymes are able to degrade arabinan in a synergistic manner. Both enzyme activities were suppressed with EDTA treatment, suggesting that they require divalent metal ions. The GH43 domains of BlArafC and BlArafB are classified into GH43 subfamilies 27 and 22, respectively, but show very low similarity (less than 15% identity) with other biochemically characterized members in the corresponding subfamilies. The B. longum subsp. longum strain lacking the GH43 gene cluster that includes BLLJ_1850 to BLLJ_1853 did not grow in arabinan medium, suggesting that BlArafC and BlArafB are important for assimilation of arabinan. IMPORTANCE We identified two novel α-l-arabinofuranosidases, BlArafC and BlArafB, from B. longum subsp. longum JCM 1217, both of which are predicted to be extracellular membrane-bound enzymes. The former specifically acts on α1,2/3-l-arabinofuranosyl linkages, while the latter acts on the α1,5-l-arabinofuranosyl linkage. These enzymes cooperatively degrade arabinan and are required for the efficient growth of bifidobacteria in arabinan-containing medium. The genes encoding these enzymes are located side by side in a gene cluster involved in metabolic pathways for plant-derived polysaccharides, which may confer adaptability in adult intestines.

Molecular determinants of substrate specificity revealed by the structure ofClostridium thermocellumarabinofuranosidase 43A from glycosyl hydrolase family 43 subfamily 16

The recent division of the large glycoside hydrolase family 43 (GH43) into subfamilies offers a renewed opportunity to develop structure–function studies aimed at clarifying the molecular determinants of substrate specificity in carbohydrate-degrading enzymes. α-L-Arabinofuranosidases (EC 3.2.1.55) remove arabinose side chains from heteropolysaccharides such as xylan and arabinan. However, there is some evidence suggesting that arabinofuranosidases are substrate-specific, being unable to display a debranching activity on different polysaccharides. Here, the structure ofClostridium thermocellumarabinofuranosidase 43A (CtAbf43A), which has been shown to act in the removal of arabinose side chains from arabinoxylan but not from pectic arabinan, is reported.CtAbf43A belongs to GH43 subfamily 16, the members of which have a restricted capacity to attack xylans. The crystal structure ofCtAbf43A comprises a five-bladed β-propeller fold typical of GH43 enzymes.CtAbf43A displays a highly compact architecture compatible with its high thermostability. Analysis ofCtAbf43A along with the other member of GH43 subfamily 16 with known structure, theBacillus subtilisarabinofuranosidase BsAXH-m2,3, suggests that the specificity of subfamily 16 for arabinoxylan is conferred by a long surface substrate-binding cleft that is complementary to the xylan backbone. The lack of a curved-shaped carbohydrate-interacting platform precludes GH43 subfamily 16 enzymes from interacting with the nonlinear arabinan scaffold and therefore from deconstructing this polysaccharide.