WrbA from Escherichia coli and Archaeoglobus fulgidus Is an NAD(P)H:Quinone Oxidoreductase

Abstract

WrbA (tryptophan [W] repressor-binding protein) was discovered inEscherichia coli, where it was proposed to play a role in regulation of the tryptophan operon; however, this has been put in question, leaving the function unknown. Here we report a phylogenetic analysis of 30 sequences which indicated that WrbA is the prototype of a distinct family of flavoproteins which exists in a diversity of cell types across all three domains of life and includes documented NAD(P)H:quinone oxidoreductases (NQOs) from the Fungi and Viridiplantae kingdoms. Biochemical characterization of the prototypic WrbA protein from E. coli and WrbA from Archaeoglobus fulgidus, a hyperthermophilic species from the Archaea domain, shows that these enzymes have NQO activity, suggesting that this activity is a defining characteristic of the WrbA family that we designate a new type of NQO (type IV). For E. coli WrbA, the K m NADH was 14 ؎ 0.43 M and the K m benzoquinone was 5.8 ؎ 0.12 M. For A. fulgidus WrbA, the K m NADH was 19 ؎ 1.7 M and the K m benzoquinone was 37 ؎ 3.6 M. Both enzymes were found to be homodimeric by gel filtration chromatography and homotetrameric by dynamic light scattering and to contain one flavin mononucleotide molecule per monomer. The NQO activity of each enzyme is retained over a broad pH range, and apparent initial velocities indicate that maximal activities are comparable to the optimum growth temperature for the respective organisms. The results are discussed and implicate WrbA in the two-electron reduction of quinones, protecting against oxidative stress.The tryptophan (W) repressor-binding protein (WrbA) from Escherichia coli (WrbA E. coli ) was discovered in 1993, when it was copurified with the tryptophan repressor (TrpR) (66). Biochemical characterization of WrbA E. coli showed that the protein binds one flavin mononucleotide (FMN) molecule per monomer and is multimeric in solution (29). These results, combined with sequence analysis and homology-based structural modeling, led to the suggestion that WrbA E. coli is the founding member of a new family of multimeric flavodoxinlike proteins. It was predicted that the WrbA family contains an ␣␤ twisted open-sheet fold characteristic of flavodoxins and a conserved insertion after strand ␤4, forming an additional ␣␤ unit (28). The presence of this fold has recently been confirmed in the published crystal structures of the Deinococcus radiodurans and Pseudomonas aeruginosa WrbA homologues (26). WrbA E. coli is the only purified and biochemically characterized WrbA protein in the literature. No enzyme activity was reported; however, it was reported that WrbA E. coli enhances the development of noncovalent complexes between the TrpR holorepressor and operator DNA and that WrbA E. coli alone is not competent to interact with the DNA targets (66). Thus, it was proposed that WrbA E. coli is an accessory element which blocks TrpR-specific transcriptional events that are deleterious to cells entering stationary phase. However, it was later concluded that WrbA E. coli does...

Reaction mechanism of azoreductases suggests convergent evolution with quinone oxidoreductases

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Functional Annotation of a Presumed Nitronate Monoxygenase Reveals a New Class of NADH:Quinone Reductases

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