The thiamin diphosphate (ThDP)-and flavin adenine dinucleotide (FAD)-dependent pyruvate oxidase from Lactobacillus plantarum catalyses the conversion of pyruvate, inorganic phosphate, and oxygen to acetyl-phosphate, carbon dioxide, and hydrogen peroxide. Central to the catalytic sequence, two reducing equivalents are transferred from the resonant carbanion/enamine forms of R-hydroxyethylThDP to the adjacent flavin cofactor over a distance of approximately 7 Å, followed by the phosphorolysis of the thereby formed acetyl-ThDP. Pre-steady-state and steady-state kinetics using time-resolved spectroscopy and a 1 H NMR-based intermediate analysis indicate that both processes are kinetically coupled. In the presence of phosphate, intercofactor electron-transfer (ET) proceeds with an apparent first-order rate constant of 78 s -1 and is kinetically gated by the preceding formation of the tetrahedral substrateThDP adduct 2-lactyl-ThDP and its decarboxylation. No transient flavin radicals are detectable in the reductive half-reaction. In contrast, when phosphate is absent, ET occurs in two discrete steps with apparent rate constants of 81 and 3 s -1 and transient formation of a flavin semiquinone/hydroxyethyl-ThDP radical pair. Temperature dependence analysis according to the Marcus theory identifies the second step, the slow radical decay to be a true ET reaction. The redox potentials of the FAD ox /FAD sq (E 1 ) -37 mV) and FAD sq /FAD red (E 2 ) -87 mV) redox couples in the absence and presence of phosphate are identical. Both the Marcus analysis and fluorescence resonance energy-transfer studies using the fluorescent N3′-pyridyl-ThDP indicate the same cofactor distance in the presence or absence of phosphate. We deduce that the exclusive 10 2 -10 3 -fold rate enhancement of the second ET step is rather due to the nucleophilic attack of phosphate on the kinetically stabilized hydroxyethyl-ThDP radical resulting in a low-potential anion radical adduct than phosphate in a docking site being part of a through-bonded ET pathway in a stepwise mechanism of ET and phosporolysis. Thus, LpPOX would constitute the first example of a radical-based phosphorolysis mechanism in biochemistry.Pyruvate oxidase from Lactobacillus plantarum (LpPOX, 1 EC 1.2.3.3) belongs to a superfamily of enzymes that utilize the cofactor thiamin diphosphate (ThDP), the biologically active derivative of vitamin B 1 . In addition to ThDP, LpPOX contains a flavin adenine dinucleotide (FAD) that is positioned at a distance of approximately 7 Å from the thiamin cofactor (Figure 1) (1).Pyruvate oxidase (POX) catalyses the conversion of pyruvate, inorganic phosphate, and oxygen to the high-energy metabolite acetyl phosphate, carbon dioxide, and hydrogen peroxide (2). Catalysis can be assumed to follow the typical Breslow mechanism of ThDP enzymes (Scheme 1). After ionization of the acidic C2-H of the thiazolium ring, pyruvate enters the active site where it reacts with the C2
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.electron transfer ͉ membrane protein ͉ X-ray crystallography R eversible binding of peripheral membrane proteins to the lipid bilayer regulates cell signaling, lipid metabolism and many other cellular events. Proteins that adhere directly to the biological membrane are termed amphitropic proteins and can attach to the bilayer through interaction of amphipathic helices, hydrophobic loops, ions, or covalently attached lipids (1, 2). In many cases studied, these proteins exhibit a very low basal membrane affinity, becoming recruited to the membrane from the cytosol only after a conformational transition or electrostatic switch that not only triggers membrane binding but may also initiate or elevate biological activity (3). Despite many recent advances in understanding how membrane binding and concomitant functional activation of proteins are regulated, there remains a paucity of structural data that allow detailed atomic insights into the nature of reversible protein-membrane interaction and of structural transitions that trigger membrane binding and functionality.In this regard, the thiamin diphosphate-(ThDP, the functional derivative of vitamin B1) and flavin-dependent pyruvate oxidase from Escherichia coli (EcPOX, EC 1.2.2.2) is a particularly interesting and extensively studied peripheral membrane protein that feeds electrons from the cytosol directly into the respiratory chain at the membrane (4-11). EcPOX supports aerobic growth in E. coli as a backup system to the pyruvate dehydrogenase multienzyme complex and catalyzes the oxidative decarboxylation of the metabolite pyruvate to carbon dioxide and acetate (12). The 2 electrons arising from oxidation of pyruvate at the ThDP site are transferred initially to the neighboring flavin (Eq.
Menaquinone (MK) is an electron carrier molecule essential for respiration in most Gram positive bacteria. A crucial step in MK biosynthesis involves the prenylation of an aromatic molecule, catalyzed by integral membrane prenyltransferases of the UbiA (4‐hydroxybenzoate oligoprenyltransferase) superfamily. In the classical MK biosynthetic pathway, the prenyltransferase responsible is MenA (1,4‐dihydroxy‐2‐naphthoate octaprenyltransferase). Recently, an alternative pathway for formation of MK, the so‐called futalosine pathway, has been described in certain micro‐organisms. Until now, five soluble enzymes (MqnA‐MqnE) have been identified in the first steps. In this study, the genes annotated as ubiA from T. thermophilus and S. lividans were cloned, expressed and investigated for prenylation activity. The integral membrane proteins possess neither UbiA nor MenA activity and represent a distinct class of prenyltransferases associated with the futalosine pathway that we term MqnP. We identify a critical residue within a highly conserved Asp‐rich motif that serves to distinguish between members of the UbiA superfamily.
The thiamine diphosphate-and flavin-dependent peripheral membrane enzyme pyruvate oxidase from Escherichia coli (EcPOX) has been crystallized in the full-length form and as a proteolytically activated C-terminal truncation variant which lacks the last 23 amino acids (Á23 EcPOX). Crystals were grown by the hanging-drop vapour-diffusion method using either protamine sulfate (fulllength EcPOX) or 2-methyl-2,4-pentanediol (Á23 EcPOX) as precipitants. Native data sets were collected at a X-ray home source to a resolution of 2.9 Å . The two forms of EcPOX crystallize in different space groups. Whereas fulllength EcPOX crystallizes in the tetragonal space group P4 3 2 1 2 with two monomers per asymmetric unit, the crystals of Á23 EcPOX belong to the orthorhombic space group P2 1 2 1 2 1 and contain 12 monomers per asymmetric unit.
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