Structural basis for membrane binding and catalytic activation of the peripheral membrane enzyme pyruvate oxidase from Escherichia coli

• Published in: Proceedings of the National Academy of Sciences - PNAS 2008-11, Vol.105 (45), p.17390-17395
• Main Authors: Neumann, Piotr, Weidner, Annett, Pech, Andreas, Stubbs, Milton T, Tittmann, Kai
• Format: Article
• Language: English
• Subjects: Active sites; Binding sites; Biochemistry; Biological Sciences; Catalysis; Catalytic oxidation; Cell Membrane - metabolism; Crystallography, X-Ray; E coli; Electrons; Enzyme Activation - genetics; Enzymes; Escherichia coli; Escherichia coli - enzymology; Lipids; Membranes; Models, Molecular; Monomers; Oxidases; P branes; Protein Conformation; Proteins; Pyruvate Oxidase - chemistry; Pyruvate Oxidase - metabolism; Pyruvic Acid - metabolism
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0805027105

The thiamin- and flavin-dependent peripheral membrane enzyme pyruvate oxidase from E. coli catalyzes the oxidative decarboxylation of the central metabolite pyruvate to CO₂ and acetate. Concomitant reduction of the enzyme-bound flavin triggers membrane binding of the C terminus and shuttling of 2 electrons to ubiquinone 8, a membrane-bound mobile carrier of the electron transport chain. Binding to the membrane in vivo or limited proteolysis in vitro stimulate the catalytic proficiency by 2 orders of magnitude. The molecular mechanisms by which membrane binding and activation are governed have remained enigmatic. Here, we present the X-ray crystal structures of the full-length enzyme and a proteolytically activated truncation variant lacking the last 23 C-terminal residues inferred as important in membrane binding. In conjunction with spectroscopic results, the structural data pinpoint a conformational rearrangement upon activation that exposes the autoinhibitory C terminus, thereby freeing the active site. In the activated enzyme, Phe-465 swings into the active site and wires both cofactors for efficient electron transfer. The isolated C terminus, which has no intrinsic helix propensity, folds into a helical structure in the presence of micelles.