Crystallization of the flavoprotein WrbA optimized by using additives and gels

Wolfova, J.; Grandori, Rita; Kozma, Erika; Chatterjee, Neal; Carey, Jannette; Smatanová, Ivana Kutá

doi:10.1016/j.jcrysgro.2005.07.043

Cited by 9 publications

(11 citation statements)

References 15 publications

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“…Aloui et al (2011) reported that S-adenosyl-Lmethionine plays an important role in anti-Cd stress in Medicago truncatula. The flavoprotein WrbA-like was derived according to its reported effect on the binding interaction between DNA and the tryptophan repressor, which regulates the biosynthesis of tryptophan in prokaryotes (Wolfova et al 2005). However, growing evidence implicates proteins of the WrbA family in the cellular response to oxidative stress (Jensen et al 2002).…”

Section: Amino Acid Metabolismmentioning

confidence: 99%

Comparative Proteomic Analysis of Differentially Expressed Proteins in Amaranthus hybridus L. Roots Under Cadmium Stress

Jin

Chen

et al. 2016

Water Air Soil Pollut

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Amaranthus hybridus L. has great potential for use in phytoremediation of soils contaminated with cadmium (Cd). In this study, we found higher absorption of Cd by the roots of A. hybridus than by its other organs. To understand the mechanism of Cd accumulation in A. hybridus roots, a comparative proteomic approach was used to differentiate the two-dimensional electrophoretic profiles of root proteins in Cd-free and Cd-treated plants. Twenty-eight differentially expressed proteins were successfully identified using matrixassisted laser desorption/ionization tandem time-offlight mass spectrometry. Of these, 10 were specifically expressed under Cd stress, and another 11 were upregulated and 7 downregulated by >2.5-fold under Cd stress. We observed increased expression of proteins involved in energy metabolism, protein metabolism, stress and defense, and signal transduction. These changes likely enhanced Cd tolerance and enrichment in A. hybridus. The downregulated proteins were mainly involved in the synthesis of microRNAs, cell walls, and other structural components. These observations were further confirmed by quantitative fluorescence PCR. The resulting differences in protein expression patterns suggest that redirection of root cell metabolism might be an important survival mechanism for A. hybridus under Cd stress.

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Section: Amino Acid Metabolismmentioning

confidence: 99%

Comparative Proteomic Analysis of Differentially Expressed Proteins in Amaranthus hybridus L. Roots Under Cadmium Stress

Jin

Chen

et al. 2016

Water Air Soil Pollut

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“…For these reasons, we have been pursuing the crystallization of E. coli WrbA. We have previously reported the crystallization of E. coli WrbA apoprotein (Wolfova et al, 2005). Here, we report the crystallization and preliminary diffraction analysis of E. coli WrbA in complex with its FMN cofactor.…”

Section: Introductionmentioning

confidence: 99%

Crystallization and preliminary diffraction analysis ofEscherichia coliWrbA in complex with its cofactor flavin mononucleotide

et al. 2007

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The flavoprotein WrbA from Escherichia coli is considered to be the prototype of a new family of multimeric flavodoxin-like proteins that are implicated in cell protection against oxidative stress. The present study is aimed at structural characterization of the E. coli protein with respect to its recently revealed oxidoreductase activity. Crystals of WrbA holoprotein in complex with the oxidized flavin cofactor (FMN) were obtained using standard vapour-diffusion techniques. Deep yellow tetragonal crystals obtained from differing crystallization conditions display different space groups and unit-cell parameters. X-ray crystal structures of the WrbA holoprotein have been determined to resolutions of 2.0 and 2.6 A.

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“…Our structures point toward mutually exclusive binding of quinones and NADH to the FMN cofactor, indicating an ordered, sequential mechanism of catalysis. Furthermore, the structures underline the obvious homologies of WrbA with the iron-sulfur flavoproteins (ISF) from Methanosarcina thermophila and A. fulgidus that form the same characteristic homotetrameric structures but differ significantly in functionally relevant, structural details (1).We note that at the time of submission of the manuscript, crystallization of E. coli WrbA and preliminary evaluation of data had been presented independently in two publications (33,34). …”

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confidence: 99%

“…We note that at the time of submission of the manuscript, crystallization of E. coli WrbA and preliminary evaluation of data had been presented independently in two publications (33,34).…”

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confidence: 99%

Crystal Structure of the NADH:Quinone Oxidoreductase WrbA from Escherichia coli

et al. 2007

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The flavoprotein WrbA, originally described as a tryptophan (W) repressor-binding protein in Escherichia coli, has recently been shown to exhibit the enzymatic activity of a NADH:quinone oxidoreductase. This finding points toward a possible role in stress response and in the maintenance of a supply of reduced quinone. We have determined the three-dimensional structure of the WrbA holoprotein from E. coli at high resolution (1.66 Å), and we observed a characteristic, tetrameric quaternary structure highly similar to the one found in the WrbA homologs of Deinococcus radiodurans and Pseudomonas aeruginosa. A similar tetramer was originally observed in an iron-sulfur flavoprotein involved in the reduction of reactive oxygen species. Together with other, recently characterized proteins such as YhdA or YLR011wp (Lot6p), these tetrameric flavoproteins may constitute a large family with diverse functions in redox catalysis. WrbA binds substrates at an active site that provides an ideal stacking environment for aromatic moieties, while providing a pocket that is structured to stabilize the ADP part of an NADH molecule in its immediate vicinity. Structures of WrbA in complex with benzoquinone and NADH suggest a sequential binding mechanism for both molecules in the catalytic cycle.In 1993, a previously unknown protein was reported to copurify with the tryptophan repressor TrpR of Escherichia coli and was, due to this assumed interaction, termed tryptophan (W) repressor binding protein, WrbA (36). At closer inspection, WrbA was found to bind a flavin mononucleotide (FMN), making it a founding member of a novel family of flavodoxins (12). Based on sequence analyses, WrbA was predicted to show the typical ␤-␣-␤-fold of flavodoxins, with a twisted, fivestranded, parallel ␤-sheet and a binding site for the FMN cofactor at the carboxy terminal end of this ␤-sheet. Distinct from the basic flavodoxin fold (30), a conserved insertion was found in sheet ␤5 (residues 140 to 160) and was predicted to form an additional ␣␤-unit (11). Proteins with this type of insertion have been classified as long-chain flavodoxins (23). The functional role of WrbA remained controversial, as initial work indicated that E. coli WrbA promotes complex formation between the repressor TrpR and its operator DNA. WrbA alone failed to bind to DNA, such that it was consequently proposed to be an accessory element that could enhance TrpRdependent repression of genes upon transition to stationary phase (36). Subsequent experiments addressed the influence of WrbA on the TrpR-DNA complex, and as these did not show any effect, the involvement of WrbA in transcription regulation was revoked (12).While the function of WrbA was entirely enigmatic, sequence database searches hinted that the WrbA family of flavoproteins was homologous to a group of several biochemically characterized NAD(P)H:quinone oxidoreductases (NQO) from various species of fungi and green plants (2,3,5,13,17,22,35). Based on subsequent biochemical assays, NQO activity was demonstrated for the WrbA...

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Crystallization of the flavoprotein WrbA optimized by using additives and gels

Cited by 9 publications

References 15 publications

Comparative Proteomic Analysis of Differentially Expressed Proteins in Amaranthus hybridus L. Roots Under Cadmium Stress

Comparative Proteomic Analysis of Differentially Expressed Proteins in Amaranthus hybridus L. Roots Under Cadmium Stress

Crystallization and preliminary diffraction analysis ofEscherichia coliWrbA in complex with its cofactor flavin mononucleotide

Crystal Structure of the NADH:Quinone Oxidoreductase WrbA from Escherichia coli

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