Crystallization and preliminary diffraction analysis of<i>Escherichia coli</i>WrbA in complex with its cofactor flavin mononucleotide

Wolfova, J.; Mesters, J.R.; Brynda, J.; Grandori, Rita; Natalello, Antonino; Carey, Jannette; Smatanová, Ivana Kutá

doi:10.1107/s1744309107026103

Cited by 6 publications

(8 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Secondary structure numbering follows the convention introduced for Nqo1 (Li et al 1995). Crystallization and diffraction analysis are described by Wolfová et al (2007). ( B ) WrbA tetramer.…”

Section: Resultsmentioning

confidence: 99%

“…Targeted drug therapies aim to exploit the naturally induced overexpression of Nqos in tumor cells to activate antitumor chemotherapeutics (Ernster 1987; Cadenas 1995; Li et al 1995). The crystal structure of oxidized tetrameric E. coli WrbA (Wolfová et al 2007) rationalizes functional distinctions with dimeric Nqos and monomeric flavodoxins and suggests a novel function for WrbAs and Nqos.…”

mentioning

confidence: 98%

See 1 more Smart Citation

WrbA bridges bacterial flavodoxins and eukaryotic NAD(P)H:quinone oxidoreductases

et al. 2007

Self Cite

View full text Add to dashboard Cite

The crystal structure of the flavodoxin-like protein WrbA with oxidized FMN bound reveals a close relationship to mammalian NAD(P)H:quinone oxidoreductase, Nqo1. Structural comparison of WrbA, flavodoxin, and Nqo1 indicates how the twisted open-sheet fold of flavodoxins is elaborated to form multimers that extend catalytic function from one-electron transfer between protein partners using FMN to two-electron reduction of xenobiotics using FAD. The structure suggests a novel physiological role for WrbA and Nqo1.

show abstract

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 98%

WrbA bridges bacterial flavodoxins and eukaryotic NAD(P)H:quinone oxidoreductases

et al. 2007

Self Cite

View full text Add to dashboard Cite

show abstract

“…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). …”

mentioning

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).…”

mentioning

confidence: 99%

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

et al. 2007

View full text Add to dashboard Cite

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...

show abstract

“…WrbA binds a flavin mononucleotide (FMN) cofactor and adopts a characteristic ␤-␣-␤ fold with a five-stranded, parallel ␤-sheet surrounded by five ␣-helices (30,31,(33)(34)(35)(36)(37)(38). Three conserved insertions, in ␤-strand 5 (loop 2), before ␣-helix 5 (loop 3), and between ␤2 and ␣2b (loop 1; contains ␣2a), supplement this core topology (31,33,35,38).…”

mentioning

confidence: 99%

WrpA Is an Atypical Flavodoxin Family Protein under Regulatory Control of the Brucella abortus General Stress Response System

et al. 2016

View full text Add to dashboard Cite

The general stress response (GSR) system of the intracellular pathogen Brucella abortus controls the transcription of approximately 100 genes in response to a range of stress cues. The core genetic regulatory components of the GSR are required for B. abortus survival under nonoptimal growth conditions in vitro and for maintenance of chronic infection in an in vivo mouse model. The functions of the majority of the genes in the GSR transcriptional regulon remain undefined. bab1_1070 is among the most highly regulated genes in this regulon: its transcription is activated 20-to 30-fold by the GSR system under oxidative conditions in vitro. We have solved crystal structures of Bab1_1070 and demonstrate that it forms a homotetrameric complex that resembles those of WrbA-type NADH:quinone oxidoreductases, which are members of the flavodoxin protein family. However, IMPORTANCEBrucella abortus is an etiological agent of brucellosis, which is among the most common zoonotic diseases worldwide. The general stress response (GSR) regulatory system of B. abortus controls the transcription of approximately 100 genes and is required for maintenance of chronic infection in a murine model; the majority of GSR-regulated genes remain uncharacterized. We present in vitro and in vivo functional and structural analyses of WrpA, whose expression is strongly induced by GSR under oxidative conditions. Though WrpA is structurally related to NADH:quinone oxidoreductases, it does not bind redox cofactors in solution, nor does it exhibit oxidoreductase activity in vitro. However, WrpA does affect spleen inflammation in a murine infection model. Our data provide evidence that WrpA forms a new functional class of WrbA/flavodoxin family proteins. Bacterial pathogens face fluctuating chemical and physical challenges in the host environment, including low pH, antimicrobial peptides, nutrient sequestration, and oxidative burst. These conditions can damage essential cellular components, including nucleic acids, proteins, and lipids. As such, bacteria encode a variety of protection mechanisms, including antioxidant enzymes, DNA damage repair systems, and efflux systems (1-4), which enable the cell to resist attacks by the host immune system (5-7).The diverse flavodoxin family, which is composed of enzymes that noncovalently bind a flavin cofactor and transfer electrons between a variety of substrates, can help to maintain cellular redox balance and confer resistance to oxidative stress (8-10). Flavodoxins and flavodoxin-like proteins are also known to promote pathogen virulence and to play a role in host infection and colonization (8,(11)(12)(13)(14)(15)(16)(17)(18), in addition to important roles in enzyme activation and metabolism (15). The flavodoxin-like protein WrbA (tryptophan [W] repressor binding protein) has been described for several microbial species, in which it is proposed to facilitate adaptation to varied chemical changes in the environment (13,(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)

show abstract

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

Cited by 6 publications

References 33 publications

WrbA bridges bacterial flavodoxins and eukaryotic NAD(P)H:quinone oxidoreductases

WrbA bridges bacterial flavodoxins and eukaryotic NAD(P)H:quinone oxidoreductases

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

WrpA Is an Atypical Flavodoxin Family Protein under Regulatory Control of the Brucella abortus General Stress Response System

Contact Info

Product

Resources

About