Embedding a feruloyl esterase active site into a thermophilic endoxylanase scaffold for the degradation of feruloylated xylans

Abstract

The structural complexity of xylan makes its complete degradation challenging. Strategies to improve its hydrolysis often requires enzyme cocktails with multiple specific activities or proteins harboring multiple catalytic domains. Here, we introduce a novel approach through the design of Xyn11_m1, a multifunctional enzyme that combines endoxylanase and feruloyl esterase activities, two catalytic functions involved in the hydrolysis of feruloylated xylans. Using the PluriZyme concept, an artificial feruloyl esterase active site was engineered into the scaffold of a thermophilic glycoside hydrolase family 10 xylanase, Xyn11, from Pseudothermotoga thermarum. Computational design, guided by protein energy landscape exploration simulations, revealed a surface cavity that could accommodate feruloyl-L-arabinose and a xylopentaose (a 5-xylose xylan polymer) bearing a single feruloyl-L-arabinose substitution on the central xylose unit. This cavity was subsequently remodeled into a serine-histidine-aspartic/glutamic acid catalytic triad with feruloyl esterase activity. Molecular dynamics simulations confirmed the stability of the engineered active site. Xyn11_m1 was successfully produced, crystallized, and characterized, and its xylanase activity at 90 °C against oat spelt xylan was comparable to that of the wild-type enzyme (713 ± 4 vs. 600 ± 8 units/mg), and it also displayed feruloyl esterase activity against methyl ferulate (140 ± 5 units/mg), a capability lacking in Xyn11. Notably, Xyn11_m1 exhibited approximately 2.5-fold greater activity compared with Xyn11 (513 ± 27 vs. 222 ± 9 units/mg) against wheat bran xylan containing ferulic acid ester linked to arabinofuranosyl residues. This dual functionality enables efficient degradation of feruloylated xylans, highlighting the potential of PluriZymes to advance biomass deconstruction technologies.

Keywords: Feruloyl esterase; PluriZyme; Protein Engineering; Xylan; Xylanase.

Comparative schematic representation of wild-type versus engineered PluriZyme activity on wheat bran. A magnified view of wheat bran showing a polymeric arabinoxylan structure with feruloylated arabinofuranose substitutions and dimeric crosslinks. On the left, the surface of the wild-type enzyme (green) is shown with its native xylanase active site circled in black. This enzyme has an activity of 222 ± 9 units/mg of wheat bran depolymerization. On the right, the engineered PluriZyme (red) displays two active sites, the native xylanase (black circle) and the newly introduced feruloyl esterase active site (blue circle), together achieving 513 ± 27 units/mg depolymerization activity. The arrows indicate cleavage sites on the xylan backbone and at the ferulic acid ester linkages.

Fig. 1

Schematic representation of the xylan polymer with ramifications. The xylan backbone is shown in green, the arabinose substitution in orange, and the ferulic acid group in blue.

Fig. 2

Computational analysis of the ligand binding site. (A) Interaction energy profile of global PELE exploration using feruloyl-L-arabinose. The distance from the ligand ester carbon to the nearest catalytic residue (E144 and E251) was calculated. (B) Ligand SASA (solvent accessible surface area) vs. binding energy for the local PELE simulation using feruloyl-L-arabinose bound to a 5-unit xylan polymer. This simulation focused on the local region of the identified binding site, in contrast to global exploration, which involved sampling the entire enzyme surface. (C) Comparison between the crystallographic conformation (pink, PDB code 7NL2) and the transient binding pocket identified by the PELE simulation (green). Structural visualization of the ligand–protein complex highlights the amino acid residues involved in ligand interactions: hydrophobic contacts (P269, L274, W320) and a hydrogen bond interaction (K331).

Fig. 3

Computational analysis of ligand binding and active site engineering. (A) Structural visualization of Xyn11_m1 (L271S, K275H and E272 (WT)) interacting with the ligand in a catalytic pose. (B) Binding energy profiles for Xyn11_m1, showing the ligand–serine distance vs. the interaction energy between the ligand and the engineered active site. Color-coded representations of the catalytic residue interactions are shown: black (all simulation frames), orange (simulation frames where at least one triad distance is satisfied), and blue (the two catalytic triad distances are satisfied). (C) Results from molecular dynamics simulations (8 replicas, 200 ns each) illustrating the stabilization of the ligand in a catalytic conformation within the engineered active sites. The percentage of poses in which the ligand achieves catalytic alignment is shown for all the tested mutants.

Fig. 4

Structural insights into Xyn11_m1. (A) Xyn11_m1 folding. The intrinsic xylanase catalytic pair (E144 and E251) is shown in stick representation in raspberry, whereas the artificial secondary esterase triad (S271, E272 and H275) is shown in stick representation in green. The trapped IPTG molecule is shown in stick representation in orange. (B) Superimposition of the two independent molecules within the asymmetric unit, chain A (blue) and chain B (green), at the artificial esterase secondary catalytic site. The catalytic triad of molecule B shows the hydrogen bonding pattern that is conserved among esterases.