Browsing by Author "Pech, Andreas"

Pyruvate oxidase from Escherichia coli (EcPOX) is a thiamin diphosphate- (ThDP) and FAD-dependent peripheral membrane protein that carries out the irreversible oxidative decarboxylation of pyruvate to acetate and carbon dioxide. Concomitant two-electron reduction of the flavin cofactor was suggested to induce a Structural rearrangement of the C-terminus triggering recruitment of the protein from the cytosol to the cell membrane, where the electrons are eventually transferred to final electron acceptor ubiquinone 8. Binding to the membrane, or alternatively, mild proteolytic digestion leads to a multifold enhancement of both the catalytic activity and substrate affinity. Recent X-ray crystallographic studies on EcPOX in the resting state and on a C-terminal truncation variant mimicking the membrane-bound activated form have fueled our understanding of the membrane-binding mechanism and concomitant catalytic activation. In the resting state, the auto-inhibitory C-terminal membrane anchor adopts a half-barrel/helix fold that occludes the active site. Upon activation, the C-terminus is expelled and becomes structurally flexible thereby freeing the active site. Circular dichroism spectroscopic analysis revealed the isolated C-terminus to be disordered, however, formation of a helical structure was observed in the presence of micelles. Limited proteolysis experiments indicate that activation of EcPOX involves at least two sequential structural transitions: the first occurring after binding of pyruvate to ThDP and the second after two-electron reduction of the flavin. (C) 2009 Elsevier B.V. All rights reserved.

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.