+ Site Statistics
+ Search Articles
+ Subscribe to Site Feeds
Most Shared
PDF Full Text
+ PDF Full Text
Request PDF Full Text
+ Follow Us
Follow on Facebook
Follow on Twitter
Follow on LinkedIn
+ Translate
+ Recently Requested

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

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

Febs Journal 277(13): 2892-2909

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 ((454)FSY(456)) 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 (Asp(452), Phe(454) and Tyr(456)) 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.

(PDF emailed within 0-6 h: $19.90)

Accession: 053740914

Download citation: RISBibTeXText

PMID: 20528921

DOI: 10.1111/j.1742-4658.2010.07705.x

Related references

Oxidation of Phe454 in the Gating Segment Inactivates Trametes multicolor Pyranose Oxidase during Substrate Turnover. Plos One 11(2): E0148108-E0148108, 2016

Pyranose oxidase, pyranose oxidase gene, novel recombinant DNA and process for producing pyranose oxidase. Official Gazette of the United States Patent & Trademark Office Patents 1206(4): 2921, Jan 27, 1998

The 'gating' residues Ile199 and Tyr326 in human monoamine oxidase B function in substrate and inhibitor recognition. Febs Journal 278(24): 4860-4869, 2012

Electrochemical characterization of the pyranose 2-oxidase variant N593C shows a complete loss of the oxidase function with full preservation of substrate (dehydrogenase) activity. Physical Chemistry Chemical Physics 18(47): 32072-32077, 2016

Importance of the first external loop for substrate recognition as revealed by chimeric Chlorella monosaccharide/H+ symporters. Febs Letters. 381(1-2): 127-130, 1996

Structural insights into substrate recognition by the Neurospora Varkud satellite ribozyme: importance of U-turns at the kissing-loop junction. Biochemistry 53(1): 258-269, 2014

Mutations of Thr169 affect substrate specificity of pyranose 2-oxidase from Trametes multicolor. Biocatalysis and Biotransformation 26(1-2): 120-127, 2008

Structural basis for substrate binding and regioselective oxidation of monosaccharides at C3 by pyranose 2-oxidase. Journal of Biological Chemistry 281(46): 35104-35115, 2006

Thermostable variants of pyranose 2-oxidase showing altered substrate selectivity for glucose and galactose. Journal of Agricultural and Food Chemistry 58(6): 3465-3471, 2010

The substrate oxidation mechanism of pyranose 2-oxidase and other related enzymes in the glucose-methanol-choline superfamily. Febs Journal 280(13): 3009-3027, 2013

Assessment of the role of an Q loop of cholesterol oxidase: a truncated loop mutant has altered substrate specificity. Biochemistry (American Chemical Society) 37(16): 70-8, 1998

Assessment of the role of OMEGA loop of cholesterol oxidase: A truncated loop mutants has altered substrate specificity. Biochemistry 37(16): 5770-5778, April 21, 1998

Assessment of the role of an omega loop of cholesterol oxidase: a truncated loop mutant has altered substrate specificity. Biochemistry 37(16): 5770-5778, 1998

Batch production of Pyranose 2-oxidase from Trametes versicolor (ATCC 11235) in medium with a lignocellulosic substrate and enzymatic bleaching of cotton fabrics. World Journal of Microbiology & Biotechnology 28(4): 1523-1531, 2013