Jul 2026· International Journal of Molecular Sciences· Vol 27· 0 citations· 61 references
Medicine
TL;DR
The high-resolution crystal structure of TsaGH11 provided a better understanding of the molecular mechanism of TsaGH11 and offer valuable guidance for the rational engineering of GH11 xylanases for industrial applications.
Abstract
Xylanases catalyze the hydrolysis of β-1,4-xylosidic linkages in xylan, a major component of plant cell walls, and are widely used in the food, feed, pulp and paper, and biofuel industries. GH11 xylanase from the hemicellulose-degrading bacterium Thermoanaerobacterium saccharolyticum (TsaGH11) exhibits high catalytic activity, making it an attractive enzyme for industrial applications. The flexibility of the thumb domain of TsaGH11 has been investigated under cryogenic and room temperature conditions; however, the substrate recognition mechanism of TsaGH11 at the optimal temperature is unknown. To better understand the molecular mechanism of substrate recognition, the high-resolution crystal structure of TsaGH11 was determined at 1.4 Å resolution. All-atom molecular dynamics simulations at 300, 320, 340, and 360 K revealed that increasing the temperature induced fluctuations in the substrate-recognizing thumb domain. At an optimal temperature of 340 K, the substrate-binding cleft of TsaGH11 predominantly adopted a closed conformation. However, the thumb domain exhibited larger fluctuations at 340 K than at other temperatures, sampling both open and closed conformations, suggesting that substrate recognition in TsaGH11 proceeds through a conformational selection-like mechanism. At 360 K, TsaGH11 unfolded partially at a site opposite the substrate-binding cleft, providing potential targets for protein engineering to improve its thermostability for industrial applications. These findings provide a better understanding of the molecular mechanism of TsaGH11 and offer valuable guidance for the rational engineering of GH11 xylanases for industrial applications.
Xylanases with high catalytic efficiency and environmental robustness are important for lignocellulosic biomass valorization, but many enzymes are rapidly inactivated under the alkaline and high-temperature conditions used in industrial processes. In this study, a computationally guided rational-design strategy was developed to improve the catalytic performance and stability of the alkaline xylanase BhS7Xyl. Constant-pH molecular dynamics, isothermal compressibility perturbation analysis, and ECNet-assisted fitness prediction were integrated to identify alkaline-sensitive and structurally unstable residues for engineering. The triple mutant H51R/D150N/E287K showed the best overall performance, with a specific activity of 1045.29 U/mg, representing a 3.73-fold increase compared with the wild type. Its melting temperature increased from 55.82 °C to 64.58 °C, while its half-life at pH 10.0 increased from 33.96 to 95.84 min. The thermal half-life at 75 °C was extended from 10.97 to 215.42 min, corresponding to a 19.64-fold improvement. Structural analyses suggested that the improved performance of H51R/D150N/E287K was associated with a more continuous xylohexaose-binding interface, increased hydrogen-bonding contacts, additional electrostatic/polar interactions, strengthened local interaction networks and enhanced dissipation of local thermal perturbation. Under optimized hydrolysis conditions, the triple mutant produced higher levels of xylose and xylooligosaccharides from standard xylan, corn cob xylan, and hardwood pulp xylan than the wild type. These work demonstrates that multi-shell electrostatic remodeling is a useful strategy for improving the activity, alkaline tolerance, and thermal stability of xylanase for xylooligosaccharide production.
Chun-Lin Tan, Xin Yu, Lanxi Sun et al.· International Journal of Bio...· 0 citations
The deacetylase repertoire is expanded and provides a framework for engineering stable industrial enzymes and molecular dynamics simulations revealed that T93P reduces backbone flexibility, C153T enhances β-sheet rigidity via hydrogen bonding, and A244M improves hydrophobic packing by filling a core cavity.
Zechang Sun, Yuxin Xia, Yiran Li et al.· Journal of Agricultural and...· 0 citations
Xylanases play important roles in lignocellulosic biomass degradation and have broad industrial applications. The discovery of new xylanases expands our understanding of these enzymes and provides new opportunities for industrial applications. This study performed biochemical and structural characterization of the xylanase Xyn11D from Hypocrea virens (HviXyn11D) and evaluated its potential for XOS production from agro-food by-products. HviXyn11D exhibited optimal hydrolytic activity against beechwood xylan at 50 °C and pH 5. Its enzymatic activity was enhanced in the presence of the biomass-derived compounds furfural, 5-hydroxymethylfurfural, and vanillin. The crystal structure of HviXyn11D exhibited a typical β-jelly roll fold and showed a unique substrate-binding cleft compared with homologous xylanases, adopting a closed conformation of the substrate-binding cleft with rigid thumb and finger domains. Molecular dynamics simulations demonstrated that higher temperatures increased the flexibility of the thumb and finger domains, leading to an open conformation of the substrate-binding cleft. Recombinant HviXyn11D enabled the hydrolysis of agro-biomass by-products derived from rice straw, corn cob, and sugarcane bagasse, resulting in successful XOS production. Together, these findings highlight the potential industrial applications of HviXyn11D in biomass degradation and provide new insights into the molecular and structural functions of HviXyn11D and the GH11 xylanase family.
Yena Kim, Bo-Min Jung, Kyeong-Min Kim et al.· International Journal of Bio...· 0 citations
This work rationally engineered flexible loops near the active site via molecular dynamics, NMsim, NMsim, sequence conservation analysis, and MAESTRO prediction, and obtained the optimal double mutant M1, which showed 1.54-fold higher specific activity and nearly 2-fold improved pH stability at pH 8, with a nearly doubled kcat while retaining substrate affinity.
Yuan Li, Yuwei Ma, Tongli Li et al.· Journal of Agricultural and...· 0 citations
This work offers the first molecular insight into P. eryngii xylanases, highlighting PeXyn1 for agro-waste valorization and multiivalent interactions at the CBM1-catalytic interface restrict structural fluctuations, promoting a stable conformation.
Xiaohang Li, Xian-Feng Du, Yan Zhang et al.· Journal of Agricultural and...· 0 citations
The industrial application of enzyme catalysts is often constrained by the trade-off between thermostability and catalytic activity. Here, a region-focused engineering strategy was applied to a thermophilic GH10 xylanase to simultaneously improve both properties. The strategy integrates qProtein-guided hydrophobic cluster design for scaffold stabilization and dynamic loop analysis for active-site optimization. The resulting triple mutant A206S-N209D-F130L exhibited substantially improved thermostability, with a 5.79 °C increase in melting temperature and an 18.8-fold extension of the half-life at 60 °C. Its optimum temperature increased from 60 to 70 °C, accompanied by a 129.4% enhancement in catalytic activity at 70 °C relatively to the wild type. Molecular dynamics simulations indicated that these mutations reshape the conformational energy landscape by stabilizing hydrophobic packing and modulating loop dynamics. This study provides a generalizable framework for simultaneously improving enzyme stability and catalytic performance.
Zhaoran Li, Zhixin Dou, Sha Zhao et al.· Journal of Agricultural and...· 0 citations
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