WrbA fromEscherichia coliandArchaeoglobus fulgidusIs an NAD(P)H:Quinone Oxidoreductase

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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 theFungiandViridiplantaekingdoms. Biochemical characterization of the prototypic WrbA protein fromE. coliand WrbA fromArchaeoglobus fulgidus, a hyperthermophilic species from theArchaeadomain, 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). ForE. coliWrbA, theKmNADHwas 14 ± 0.43 μM and theKmbenzoquinonewas 5.8 ± 0.12 μM. ForA. fulgidusWrbA, theKmNADHwas 19 ± 1.7 μM and theKmbenzoquinonewas 37 ± 3.6 μM. 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.