5HXV

The crystal structure of thermostable xylanase mutant

5HXV の概要

• エントリーDOI: 10.2210/pdb5hxv/pdb
• 分子名称: Endo-1,4-beta-xylanase (2 entities in total)
• 機能のキーワード: glycoside hydrolase family 11 endo-xylanase, hydrolase
• 由来する生物種: Talaromyces cellulolyticus CF-2612
• タンパク質・核酸の鎖数: 12
• 化学式量合計: 247491.91

構造登録者

Watanabe, M.,Ishikawa, K. (登録日: 2016-01-31, 公開日: 2016-07-27, 最終更新日: 2024-11-13)

主引用文献

Watanabe, M.,Fukada, H.,Ishikawa, K.
Construction of Thermophilic Xylanase and Its Structural Analysis
Biochemistry, 55:4399-4409, 2016

PubMed Abstract: The glycoside hydrolase family 11 xylanase has been utilized in a wide variety of industrial applications, from food processing to kraft pulp bleaching. Thermostability enhances the economic value of industrial enzymes by making them more robust. Recently, we determined the crystal structure of an endo-β-1,4-xylanase (GH11) from mesophilic Talaromyces cellulolyticus, named XylC. Ligand-free XylC exists to two conformations (open and closed forms). We found that the "closed" structure possessed an unstable region within the N-terminal region far from the active site. In this study, we designed the thermostable xylanase by the structure-based site-directed mutagenesis on the N-terminal region. In total, nine mutations (S35C, N44H, Y61M, T62C, N63L, D65P, N66G, T101P, and S102N) and an introduced disulfide bond of the enzyme contributed to the improvement in thermostability. By combining the mutations, we succeeded in constructing a mutant for which the melting temperature was partially additively increased by >20 °C (measured by differential scanning calorimetry) and the activity was additively enhanced at elevated temperatures, without loss of the original specific activity. The crystal structure of the most thermostable mutant was determined at 2.0 Å resolution to elucidate the structural basis of thermostability. From the crystal structure of the mutant, it was revealed that the formation of a disulfide bond induces new C-C contacts and a conformational change in the N-terminus. The resulting induced conformational change in the N-terminus is key for stabilizing this region and for constructing thermostable mutants without compromising the activity.

PubMed: 27410423
DOI: 10.1021/acs.biochem.6b00414

実験手法

X-RAY DIFFRACTION (2 Å)