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

Abstract

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...

Compensations for Diminished Terminal Oxidase Activity in Escherichia coli

“…Along with the electroneutral AppBC complex, WrbA could provide an electron sink in exponentially growing cydB cells. Unlike the membranespanning NADH dehydrogenase (NDH-1), the cytoplasmic WrbA is unlikely to translocate protons (40).…”

“…2). This illustrates that loss of cytochrome bd-I elicits the up-regulation of systems involved in the electroneutral oxidation of NADH and ubiquinol via WrbA and AppBC, respectively (WrbA is a soluble NADH-quinone oxidoreductase (40), unlikely to translocate protons due to the lack of transmembrane helices). Additionally, the up-regulation of poxB (pyruvate oxidase) and the glyoxylate shunt gene aceB suggests that the cydB cells may produce less NADH than the wild type strain.…”

KefF, the Regulatory Subunit of the Potassium Efflux System KefC, Shows Quinone Oxidoreductase Activity

“…A phenylalanine in this position is also conserved in MdaB and AzoR from E. coli (1,20). In E. coli WrbA, W67 fulfills the function, and a stacked structure with benzoquinone has been resolved (2,5).…”

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

“…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).…”

“…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). Insertion loop 1 distinguishes WrbA from classic flavodoxins (15).…”