A Molecular Switch and Proton Wire Synchronize the Active Sites in Thiamine Enzymes

Frank, René; Titman, Christopher M.; Pratap, J. Venkatesh; Luisi, Ben F.; Perham, Richard N.

doi:10.1126/science.1101030

Cited by 172 publications

(203 citation statements)

References 33 publications

Supporting

Mentioning

186

Contrasting

Order By: Relevance

“…There is no structural non-equivalence of the monomers detectable as observed for pyruvate dehydrogenase E1 component, for which a half-of-sites mechanism has been suggested ( Fig. S2) (24,25). The subunit structure of EcPOX (Fig.…”

Section: Resultsmentioning

confidence: 99%

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

Neumann

Weidner

Pech

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

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.

show abstract

Section: Resultsmentioning

confidence: 99%

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

Neumann

Weidner

Pech

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…The major conclusion derived from structural, kinetic, and spectroscopic studies using substitutions at the Glu 235 , Glu 237 , and Arg 606 positions, residues that along with the highly conserved Glu 571 residue are hypothesized to form a communication pathway between the two active center ThDPs (2,3), is that ThDP binding in the second active center is affected by these substitutions according to the following observations. 1) Steady-state kinetic analysis revealed that ThDP binding is affected.…”

Section: Discussionmentioning

confidence: 99%

“…Communication between the thiamin diphosphate cofactors (ThDPs) 4 at the active centers of ThDP enzymes has been reported for several years (1)(2)(3)(4)(5)(6)). An intriguing pathway for such interaction had been suggested by Perham's group (2) on the basis of crystal structure studies on E1bs (the E1 component of the pyruvate dehydrogenase complex (PDHc) from Bacillus stearothermophilus).…”

mentioning

confidence: 99%

“…An intriguing pathway for such interaction had been suggested by Perham's group (2) on the basis of crystal structure studies on E1bs (the E1 component of the pyruvate dehydrogenase complex (PDHc) from Bacillus stearothermophilus). According to this suggestion, there is a hydrated tunnel of acidic residues involved in proton shuttling from the N1Ј atom of the ThDP in one active site (activated ThDP, active site is closed) to the corresponding atom in the second active site (non-activated ThDP, active site is open) over a distance of 20 Å, dubbed a "proton wire" (2). This mechanism explains how two active sites communicate with each other, with no significant conformational changes in the structure of the subunits with the exception of the active site loops (2).…”

mentioning

confidence: 99%

“…According to this suggestion, there is a hydrated tunnel of acidic residues involved in proton shuttling from the N1Ј atom of the ThDP in one active site (activated ThDP, active site is closed) to the corresponding atom in the second active site (non-activated ThDP, active site is open) over a distance of 20 Å, dubbed a "proton wire" (2). This mechanism explains how two active sites communicate with each other, with no significant conformational changes in the structure of the subunits with the exception of the active site loops (2). At the same time, for E1h (human PDHc E1), a so-called "flip-flop" mechanism was proposed for active site communication through the shuttle-like motion of the heterotetramers, although the x-ray structure did not reveal directly a structural nonequivalence of the two active centers (4).…”

mentioning

confidence: 99%

See 2 more Smart Citations