Molecular Basis of Arabinobio-hydrolase Activity in Phytopathogenic Fungi

Abstract

The phytopathogenic fungus Fusarium graminearum secretes a very diverse pool of glycoside hydrolases (GHs) aimed at degrading plant cell walls. ␣-L-Arabinanases are essential GHs participating in the complete hydrolysis of hemicellulose, a natural resource for various industrial processes, such as bioethanol or pharmaceuticals production. Arb93A, the exo-1,5-␣-L-arabinanase of F. graminearum encoded by the gene fg03054.1, belongs to the GH93 family, for which no structural data exists. The enzyme is highly active (1065 units/mg) and displays a strict substrate specificity for linear ␣-1,5-L-arabinan. Biochemical assays and NMR experiments demonstrated that the enzyme releases ␣-1,5-L-arabinobiose from the nonreducing end of the polysaccharide. We determined the crystal structure of the native enzyme and its complex with ␣-1,5-L-arabinobiose, a degradation product of ␣-Me-1,5-L-arabinotetraose, at 1.85 and 2.05 Å resolution, respectively. Arb93A is a monomeric enzyme, which presents the six-bladed ␤-propeller fold characteristic of sialidases of clan GHE. The configuration of the bound arabinobiose is consistent with the retaining mechanism proposed for the GH93 family. Catalytic residues were proposed from the structural analysis, and site-directed mutagenesis was used to validate their role. They are significantly different from those observed for GHE sialidases.The plant cell wall consists mainly of a complex aggregation of polysaccharides, such as cellulose, hemicellulose, and pectin. Hemicellulose is one of the most abundant renewable biopolymers on earth and constitutes an important source of energy for the biofuel industry. It represents 20 -40% of plant biomass and is principally composed of pentoses, such as xylose and arabinose (1). Due to the high complexity and structural variability of this polysaccharide, many enzymes are necessary for its complete degradation (2). A number of microorganisms are able to break down hemicellulose, through the action of various glycoside hydrolases (GHs).2 The latter catalyze the cleavage of glycosidic bonds between sugars with either inversion or retention of the anomeric configuration (3). GHs have been classified into more than 114 different families based on their amino acid sequence similarity (CAZY (Carbohydrate Active Enzymes) server, available on the World Wide Web) (4, 5).␣-L-Arabinanases (EC 3.2.1.-) are accessory hemicellulases that hydrolyze ␣-L-arabinofuranosic linkages and act synergistically with other GHs to break down hemicellulose fully (6). These enzymes have become of interest in recent years because of their potential rate-limiting role in the degradation of lignocelluloses and their practical application in various industrial processes, such as the production of important medicinal compounds, the improvement of wine flavors, pulp treatment, juice clarification, the production of bioethanol, and the synthesis of oligosaccharides (7). According to the CAZY classification, ␣-Larabinanases are present in six GH families (3,43,51,54, 62, and 93) whose ...

Genomics Review of Holocellulose Deconstruction by Aspergilli

“…GH93 arabinanases recognize linear ␣-1,5-L-arabinan as the substrate and release ␣-1,5-L-arabinobiose from the nonreducing end of the polysaccharide (81). Two fungal GH93 arabinanases (81,184) show a six-blade ␤-propeller fold with a typical "Velcro ring" closure.…”

“…Two fungal GH93 arabinanases (81,184) show a six-blade ␤-propeller fold with a typical "Velcro ring" closure. The substrate-binding groove is enclosed at one end by two residues, Glu and Tyr, which contribute to the recognition of the nonreducing chain end of the polysaccharide (184).…”

Elucidation of the Molecular Basis for Arabinoxylan-Debranching Activity of a Thermostable Family GH62 α-l-Arabinofuranosidase from Streptomyces thermoviolaceus

“…For example, the catalytic domain of enzymes belonging to clan GH-A adopt a (␤/␣) 8 triosephosphate isomerase (TIM) barrel fold, as exemplified by the structure of the GH51 ␣-L-arabinofuranosidase from Geobacillus stearothermophilus T-6 (16). In contrast, clan GH-E enzymes adopt a six-bladed ␤-propeller architecture (15), GH43 enzymes in clan GH-F display a "non-Velcroed" five-bladed ␤-propeller arrangement (17,18), and the catalytic domain of the GH54 ␣-L-arabinofuranosidase from Aspergillus kawachii adopts a ␤-sandwich fold (19).…”

“…Moreover, family GH1 includes exo-acting arabinofuranosidases active on p-nitrophenyl-␣-arabinofuranoside (pNP-AraF) and ␣-1,5 arabino-oligosaccharides (13), family GH30 includes enzymes with 1,5-␣-L-arabinobiose activities (14), and family GH93 includes exo-acting enzymes that release arabinobiose from the nonreducing end of ␣-1,5-L-arabinan (15). Several of these enzyme families are further grouped into glycoside hydrolase clans GH-A (GH1, GH30, and GH51), GH-E (GH93), and GH-F (GH43 and GH62), reflecting the structural diversity of ␣-L-arabinofuranosidases.…”

Investigating the Function of an Arabinan Utilization Locus Isolated from a Termite Gut Community

“…In addition, exo-␣-L-1,5-arabinanase (EC 3.2.1.-) has been described in the family GH93. These latter enzymes release arabinobiose as a major product from linear arabinan (11,12). In the genomes of biomass-degrading bacteria, notably those present in gut environments, GH-encoding sequences are often grouped together in loci that are dedicated to the breakdown of specific polysaccharides (13).…”