Importance of the gating segment in the substrate-recognition loop of pyranose 2-oxidase

• Published in: The FEBS journal 2010-07, Vol.277 (13), p.2892-2909
• Main Authors: Spadiut, Oliver, Tan, Tien-Chye, Pisanelli, Ines, Haltrich, Dietmar, Divne, Christina
• Format: Article
• Language: English
• Subjects: active-site loop; alanine-scanning mutagenesis; Binding sites; Biochemistry; Biokemi; Carbohydrate Dehydrogenases - chemistry; Carbohydrate Dehydrogenases - genetics; Carbohydrate Dehydrogenases - metabolism; Catalytic Domain; Chemistry; crystal structure; Crystallography, X-Ray; Enzymes; Fungi; Kemi; Ketoses - chemical synthesis; Ketoses - chemistry; Kinetics; Lignin - chemistry; Medicin och hälsovetenskap; Models, Molecular; Molecular Structure; NATURAL SCIENCES; NATURVETENSKAP; pyranose 2-oxidase; site-saturation mutagenesis; Substrate Specificity; Substrates; Trametes - enzymology; Trametes multicolor
• ISSN: 1742-464X; 1742-4658; 1742-4658
• DOI: 10.1111/j.1742-4658.2010.07705.x

Pyranose 2-oxidase from Trametes multicolor is a 270 kDa homotetrameric enzyme that participates in lignocellulose degradation by wood-rotting fungi and oxidizes a variety of aldopyranoses present in lignocellulose to 2-ketoaldoses. The active site in pyranose 2-oxidase is gated by a highly conserved, conformationally degenerate loop (residues 450-461), with a conformer ensemble that can accommodate efficient binding of both electron-donor substrate (sugar) and electron-acceptor substrate (oxygen or quinone compounds) relevant to the sequential reductive and oxidative half-reactions, respectively. To investigate the importance of individual residues in this loop, a systematic mutagenesis approach was used, including alanine-scanning, site-saturation and deletion mutagenesis, and selected variants were characterized by biochemical and crystal-structure analyses. We show that the gating segment (⁴⁵⁴FSY⁴⁵⁶) of this loop is particularly important for substrate specificity, discrimination of sugar substrates, turnover half-life and resistance to thermal unfolding, and that three conserved residues (Asp⁴⁵², Phe⁴⁵⁴ and Tyr⁴⁵⁶) are essentially intolerant to substitution. We furthermore propose that the gating segment is of specific importance for the oxidative half-reaction of pyranose 2-oxidase when oxygen is the electron acceptor. Although the position and orientation of the slow substrate 2-deoxy-2-fluoro-glucose when bound in the active site of pyranose 2-oxidase variants is identical to that observed earlier, the substrate-recognition loop in F454N and Y456W displays a high degree of conformational disorder. The present study also lends support to the hypothesis that 1,4-benzoquinone is a physiologically relevant alternative electron acceptor in the oxidative half-reaction.