Crystallization and preliminary X-ray diffraction analysis of full-length and proteolytically activated pyruvate oxidase from<i>Escherichia coli</i>

Weidner, Annett; Neumann, Piotr; Wille, Georg; Stubbs, Milton T.; Tittmann, Kai

doi:10.1107/s1744309108003473

Cited by 6 publications

(3 citation statements)

References 19 publications

(10 reference statements)

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“…Crystals of full-length and EcPOX⌬23 were grown as detailed in ref. 44. A redundant dataset of a single EcPOX crystal was collected in-house in a 100 K nitrogen cryostream (XSTREAM2000; Rigaku/MSC) after gradually transferring the crystal into a cryoprotectant containing mother liquor supplemented with 5%, 15%, and 30% (vol/vol) glycerol.…”

Section: Methodsmentioning

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.

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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.

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Section: Methodsmentioning

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.

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“…Both the N‐ and C‐terminal ends of PoxB are critical for function, making it ineligible for mf ‐LON degradation (Neumann et al , ; Weidner et al , ). Therefore, the Potyvirus SuMMV protease was selected, which cleaves peptide bonds immediately after the sequence EEIHLQ (Fig E; Fernandez‐Rodriguez & Voigt, ).…”

Section: Resultsmentioning

confidence: 99%

Dynamic control of endogenous metabolism with combinatorial logic circuits

Moser

Borujeni

Ghodasara

et al. 2018

Molecular Systems Biology

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Controlling gene expression during a bioprocess enables real‐time metabolic control, coordinated cellular responses, and staging order‐of‐operations. Achieving this with small molecule inducers is impractical at scale and dynamic circuits are difficult to design. Here, we show that the same set of sensors can be integrated by different combinatorial logic circuits to vary when genes are turned on and off during growth. Three Escherichia coli sensors that respond to the consumption of feedstock (glucose), dissolved oxygen, and by‐product accumulation (acetate) are constructed and optimized. By integrating these sensors, logic circuits implement temporal control over an 18‐h period. The circuit outputs are used to regulate endogenous enzymes at the transcriptional and post‐translational level using CRISPRi and targeted proteolysis, respectively. As a demonstration, two circuits are designed to control acetate production by matching their dynamics to when endogenous genes are expressed (pta or poxB) and respond by turning off the corresponding gene. This work demonstrates how simple circuits can be implemented to enable customizable dynamic gene regulation.

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“…Depending on the redox‐state of the FAD, an unusual lipid‐binding domain changes its fold and, thereby, regulates the peripheral membrane association [25]. Our combined approach of electrochemistry and spectroscopy reveals details of the underlying structural transitions during the interconversion between three distinct protein conformers of the enzyme's current activation model, which has emerged from extensive biochemical [35] and structural studies [31,36]:…”

Section: Discussionmentioning

confidence: 99%

The cysteine S–H stretching mode reports on long‐range conformational changes in pyruvate oxidase from E. coli

et al. 2023

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The pyruvate oxidases from Escherichia coli (EcPOX) and Lactobacillus plantarum (LpPOX) are both thiamin‐dependent flavoenzymes. Their sequence and structure are closely related, and they catalyse similar reactions—but they differ in their activity pattern: LpPOX is always highly active, EcPOX only when activated by lipids or limited proteolysis, both involving the protein's C‐terminal 23 residues (the ‘α‐peptide’). Here, we relate the redox‐induced infrared (IR) difference spectrum of EcPOX to its unusual activation mechanism. The IR difference spectrum of EcPOX is marked by contributions from the protein backbone, reflecting major conformational changes. A rare sulfhydryl (−SH) difference signal indicates changes in the vicinity of cysteines. We could pin the Cys–SH difference signal to Cys88 and Cys494, both being remote from the moving α‐peptide and the redox‐active flavin cofactor. Yet, when the α‐peptide is proteolytically removed, the Cys–SH difference signal disappears, together with several difference signals in the amide range. The remaining IR signature of the permanently activated EcPOXΔ23 is strikingly similar to the simpler signature of LpPOX. The loss of the α‐peptide ‘transforms’ the catalytically complex EcPOX into the catalytically ‘simpler’ LpPOX.

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Crystallization and preliminary X-ray diffraction analysis of full-length and proteolytically activated pyruvate oxidase fromEscherichia coli

Cited by 6 publications

References 19 publications

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

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

Dynamic control of endogenous metabolism with combinatorial logic circuits

The cysteine S–H stretching mode reports on long‐range conformational changes in pyruvate oxidase from E. coli

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