Crystal Structure and Substrate Specificity Modification of Acetyl Xylan Esterase from Aspergillus luchuensis

Abstract

Acetyl xylan esterase (AXE) catalyzes the hydrolysis of the acetyl bonds present in plant cell wall polysaccharides. Here, we determined the crystal structure of AXE from (AXEA), providing the three-dimensional structure of an enzyme in the Esterase_phb family. AXEA shares its core α/β-hydrolase fold structure with esterases in other families, but it has an extended central β-sheet at both its ends and an extra loop. Structural comparison with a ferulic acid esterase (FAE) from indicated that AXEA has a conserved catalytic machinery: a catalytic triad (Ser119, His259, and Asp202) and an oxyanion hole (Cys40 and Ser120). Near the catalytic triad of lAXEA, two aromatic residues (Tyr39 and Trp160) form small pockets at both sides. Homology models of fungal FAEs in the same Esterase_phb family have wide pockets at the corresponding sites because they have residues with smaller side chains (Pro, Ser, and Gly). Mutants with site-directed mutations at Tyr39 showed a substrate specificity similar to that of the wild-type enzyme, whereas those with mutations at Trp160 acquired an expanded substrate specificity. Interestingly, the Trp160 mutants acquired weak but significant type B-like FAE activity. Moreover, the engineered enzymes exhibited ferulic acid-releasing activity from wheat arabinoxylan. Hemicelluloses in the plant cell wall are often decorated by acetyl and ferulic acid groups. Therefore, complete and efficient degradation of plant polysaccharides requires the enzymes for cleaving the side chains of the polymer. Since the Esterase_phb family contains a wide array of fungal FAEs and AXEs from fungi and bacteria, our study will provide a structural basis for the molecular mechanism of these industrially relevant enzymes in biopolymer degradation. The structure of the Esterase_phb family also provides information for bacterial polyhydroxyalkanoate depolymerases that are involved in biodegradation of thermoplastic polymers.

Integrated Hydrolysis of Mixed Agro-Waste for a Second Generation Biorefinery Using Nepenthes mirabilis Pod Digestive Fluids

“…Recent studies have shown carboxylesterase activity in Penicillium chrysogenum, whereby the activity was found to be 5.4 U/L [54], with Aspergillus and Trichoderma sp. being other organisms that were reported for carboxylesterase activity [55]. This illustrated the capability of the N. mirabilis pods' digestive fluids to hydrolyse acetyl groups including those associated with xylan.…”

“…Furthermore, Bacillus sp., in particular, B. vallismortis, was confirmed to have the ability to produce short-chained acetyl xylan esterases [56]. The decoupling of the xylan backbone is highly dependent on the endo/β-xylosidases; albeit acetyl xylan esterases, including xylanases, are largely involved [55]. By reducing the acetyl groups, the endo-xylanases will, thus, be effective during the hydrolysis of mixed agro-waste [30], furthering the decomposition of xylan to xylose.…”

The Synthetic Potential of Fungal Feruloyl Esterases: A Correlation with Current Classification Systems and Predicted Structural Properties

“…Homology modeling was performed based on a template structure with high sequence identity. In particular, the tannase-related SF1-3 FAEs showed high homology with the FAE B (AoFaeB) from Aspergillus oryzae [33] while SF6 acetyl xylan related-FAEs showed homology with an acetyl xylan esterase from Aspergillus luchuensis [34]. The SF5 acetyl-xylan-related FAEs showed no significant homology with determined structures (<30%), therefore, this intriguing group was not used for the prediction of surface properties and SMD simulations.…”

Characterization of new fungal carbohydrate esterase family 1 proteins leads to the discovery of two novel dual feruloyl/acetyl xylan esterases

“…It is distantly related to the other fungal CE1 members (Esterase, PHB depolymerase, IPR010126), but related to the Esterase D/S-formylglutathione hydrolase sequences (IPR014186) according to InterPro classification [22]. In comparison with bacterial CE1 enzymes, fungal CE1 enzymes mainly belong to the Esterase_phb family based on the ESTHER database, a classification for proteins with an α/β-hydrolase fold [23], which is distantly related to bacterial enzymes belonging to Antigen85c family (<20% sequence identity) [24]. Thus far, two crystal structures of fungal CE1 enzymes have been reported, i.e.…”

“…Anaeromyces mucronatus AmCE1 (PDB: 5CXU [25]) and Aspergillus luchuensis AlAXEA (formerly Aspergillus awamori) (PDB: 5X6S [24]).…”