5ABN

CRYSTAL STRUCTURE ANALYSIS OF FUNGAL VERSATILE PEROXIDASE FROM PLEUROTUS ERYNGII. MUTANT VPi. MUTATED RESIDUES D69S, T70D, S86E, D146T, Q202L, H232E, Q239R AND S301K.

5ABN の概要

• エントリーDOI: 10.2210/pdb5abn/pdb
• 関連するPDBエントリー: 5ABO 5ABQ
• 分子名称: VERSATILE PEROXIDASE VPL2, CALCIUM ION, PROTOPORPHYRIN IX CONTAINING FE, ... (5 entities in total)
• 機能のキーワード: class ii (fungal) peroxidases, protoporphyrin ix, electron transfer, lignin peroxidase, lignin degradation, manganese peroxidase, mn-independent oxidation phenolic non-phenolic aromatics, mnii oxidation, peroxidase, polyvalent peroxidase, oxidoreductase, heme, hydrogen peroxide, iron, manganese, metal-binding, secreted, zymogen
• 由来する生物種: PLEUROTUS ERYNGII (BOLETUS OF THE STEPPES)
• 細胞内の位置: Secreted : O94753
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 35412.14

構造登録者

Medrano, F.J.,Romero, A. (登録日: 2015-08-07, 公開日: 2015-11-04, 最終更新日: 2024-11-13)

主引用文献

Saez-Jimenez, V.,Fernendez-Fueyo, E.,Medrano, F.J.,Romero, A.,Martinez, A.T.,Ruiz-Duenas, F.J.
Improving the Ph-Stability of Versatile Peroxidase by Comparative Structural Analysis with a Naturally-Stable Manganese Peroxidase.
Plos One, 10:40984-, 2015

PubMed Abstract: Versatile peroxidase (VP) from the white-rot fungus Pleurotus eryngii is a high redox potential peroxidase of biotechnological interest able to oxidize a wide range of recalcitrant substrates including lignin, phenolic and non-phenolic aromatic compounds and dyes. However, the relatively low stability towards pH of this and other fungal peroxidases is a drawback for their industrial application. A strategy based on the comparative analysis of the crystal structures of VP and the highly pH-stable manganese peroxidase (MnP4) from Pleurotus ostreatus was followed to improve the VP pH stability. Several interactions, including hydrogen bonds and salt bridges, and charged residues exposed to the solvent were identified as putatively contributing to the pH stability of MnP4. The eight amino acid residues responsible for these interactions and seven surface basic residues were introduced into VP by directed mutagenesis. Furthermore, two cysteines were also included to explore the effect of an extra disulfide bond stabilizing the distal Ca2+ region. Three of the four designed variants were crystallized and new interactions were confirmed, being correlated with the observed improvement in pH stability. The extra hydrogen bonds and salt bridges stabilized the heme pocket at acidic and neutral pH as revealed by UV-visible spectroscopy. They led to a VP variant that retained a significant percentage of the initial activity at both pH 3.5 (61% after 24 h) and pH 7 (55% after 120 h) compared with the native enzyme, which was almost completely inactivated. The introduction of extra solvent-exposed basic residues and an additional disulfide bond into the above variant further improved the stability at acidic pH (85% residual activity at pH 3.5 after 24 h when introduced separately, and 64% at pH 3 when introduced together). The analysis of the results provides a rational explanation to the pH stability improvement achieved.

PubMed: 26496708
DOI: 10.1371/JOURNAL.PONE.0140984

実験手法

X-RAY DIFFRACTION (2.194 Å)