THE VERY HIGH RESOLUTION CRYSTAL STRUCTURE OF GLUCOSE OXIDASE FROM P. AMAGASAKIENSE. 1200 RESIDUES AT 1.79 Å RESOLUTION. Jörg Hendle1, Hans-Jürgen Hecht2, Henryk M. Kalisz3 & Dietmar Schomburg2. 1Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA, USA; Departments of 2Molecular Structure Research and 3Enzyme Technology, Gesellschaft für Biotechnologische Forschung, Braunschweig, Germany
Glucose oxidase, a highly glycosylated flavo protein, is one of the most widely used enzymes in medical diagnostics and food processing. The enormous economic significance and intensive application of glucose oxidase as biosensor enzyme, as integral part of food production and in fermentation control was contradicted so far by the lack of structural knowledge.
The enzyme is a homodimer of 155 kDa with a glycan moiety of the `high mannose' type representing 13% of the molecular weight. Crystals suitable for X-ray diffraction were obtained only after enzymatic deglycosylation removing 95% of the saccharide residues. As confirmed by the high resolution structure the monomer of the deglycosylated protein comprises 587 amino acid residues, 5 N-acetyl glucosamine, 3 mannose and 1 FAD molecule.
The exceptional stability of glucose oxidase might be based on the large surface area buried upon dimerization and defined by (i) hydrophobic contacts, (ii) extensive salt bridges and, surprisingly, (iii) by a cluster of well ordered water molecules trapped between the monomers with no contact to the bulk. The 67 kDa monomer is comprised of two domains only. The FAD binding domain is separated from the substrate binding domain by a deep cavity filled with a well defined network of 10 water molecules. At the given resolution this water cluster might suit as a starting point for the modeling of the substrate glucose bound to the binary enzyme:FAD complex.
The high resolution structure of glucose oxidase facilitates the use of protein engineering with the goal to design highly active enzyme derivatives with the capability to transfer directly electrons to semi-conductors and/or increased tolerance against hydrogen peroxide, sulfur dioxide and hydrogen sulfite.