1.8 and 1.9 Å resolution structures of the ­Penicillium amagasakiense and Aspergillus niger glucose oxidases as a basis for modelling substrate complexes

• Published in: Acta crystallographica. Section D, Biological crystallography. Vol. 55; no. 5; pp. 969 - 977
• Main Authors: Wohlfahrt, Gerd, Witt, Susanne, Hendle, Jörg, Schomburg, Dietmar, Kalisz, Henryk M., Hecht, Hans-Jürgen
• Format: Journal Article
• Language: English
• Published: 5 Abbey Square, Chester, Cheshire CH1 2HU, England International Union of Crystallography 01.05.1999
• Subjects: Amino Acid Sequence; Aspergillus niger - enzymology; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Crystallography, X-Ray; Flavin-Adenine Dinucleotide - chemistry; Flavin-Adenine Dinucleotide - metabolism; Glucose oxidase; Glucose Oxidase - chemistry; Glucose Oxidase - metabolism; Kinetics; Models, Molecular; Molecular Sequence Data; Monosaccharides - chemistry; Monosaccharides - metabolism; Oxidation-Reduction; Oxidoreductases; Penicillium - enzymology; Protein Conformation; Substrate Specificity; Substrate-complex modelling
• ISSN: 1399-0047; 0907-4449; 1399-0047
• DOI: 10.1107/S0907444999003431

Glucose oxidase is a flavin‐dependent enzyme which catalyses the oxidation of β‐d‐glucose by molecular oxygen to δ‐­gluconolactone and hydrogen peroxide. The structure of the enzyme from Aspergillus niger, previously refined at 2.3 Å resolution, has been refined at 1.9 Å resolution to an R value of 19.0%, and the structure of the enzyme from Penicillium amagasakiense, which has 65% sequence identity, has been determined by molecular replacement and refined at 1.8 Å resolution to an R value of 16.4%. The structures of the partially deglycosylated enzymes have an r.m.s. deviation of 0.7 Å for main‐chain atoms and show four N‐glycosylation sites, with an extended carbohydrate moiety at Asn89. Substrate complexes of the enzyme from A. niger were modelled by force‐field methods. The resulting model is consistent with results from site‐directed mutagenesis experiments and shows the β‐d‐glucose molecule in the active site of glucose oxidase, stabilized by 12 hydrogen bonds and by hydrophobic contacts to three neighbouring aromatic residues and to flavin adenine dinucleotide. Other hexoses, such as α‐­d‐­glucose, mannose and galactose, which are poor substrates for the enzyme, and 2‐deoxy‐d‐glucose, form either fewer bonds or unfavourable contacts with neighbouring amino acids. Simulation of the complex between the reduced enzyme and the product, δ‐gluconolactone, has provided an explanation for the lack of product inhibition by the lactone.