WrbA from Escherichia coli and Archaeoglobus fulgidus is an NAD(P)H:quinone oxidoreductase

Abstract

WrbA (tryptophan [W] repressor-binding protein) was discovered in Escherichia 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 microM and the K(m)(benzoquinone) was 5.8 +/- 0.12 microM. For A. fulgidus WrbA, the K(m)(NADH) was 19 +/- 1.7 microM and the K(m)(benzoquinone) was 37 +/- 3.6 microM. 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.

FIG. 1.

Alignment of WrbA, NAD(P)H:quinone oxidoreductase, and flavodoxin sequences. Numerical values at the C terminus indicate percent identity/percent similarity to WrbA_{E. coli}. ns, not significant; #, completely conserved residue. Shaded residues indicate that a conservation of similar residues persists across at least 60% of the alignment. = = = = = = = =, region of the flavodoxin signature motif. JPred at ExPASy's Proteomic Tools was used to predict α-helix (HHHH) and β-sheet (SSSS) sequences with the alignment (flavodoxin sequences were excluded). The additional αβ unit that is discussed is indicated in bold below the secondary-structure prediction. Organisms and ORF designations from the corresponding genomic sequence: A. fulgidus WrbA (gi: 11497955), A. thaliana FQR1 (gi: 21539481), Burkholderia cepacia WrbA (gi: 46310790), Chloroflexus aurantiacus WrbA (gi: 53798190), Caulobacter crescentus WrbA (gi: 13422034), C. beijerinckii Fldx (gi: 1941945), Desulfovibrio gigas Fldx (gi: 40801), D. radiodurans WrbA (gi: 58177611), Exiguobacterium sp. strain 255-15 WrbA (gi: 68054352), Erwinia carotovora WrbA (gi: 49613050), E. coli WrbA (gi: 148264), G. trabeum QR1 (gi: 30027749) and QR2 (gi: 33668045), Methanosarcina acetivorans WrbA (gi: 19915027), Mycobacterium avium WrbA (gi: 41409133), Methanosarcina barkeri WrbA (gi: 31793946) and Fldx (gi: 72396729), Mezorhizobium sp. strain BNC1 WrbA (gi: 68191386), Microbulbifer degredans WrbA (gi: 48860599), Methanosarcina mazei WrbA (gi: 21228326), Neurospora crassa Hyp (gi: 38566940), Nostoc punctiforme WrbA (gi: 23129682), Psychrobacter arcticum WrbA(gi: 71038102), P. chrysosporium QR (gi: 4454993), Polaromonas sp. strain JS666 WrbA (gi: 67847813), P. aeruginosa WrbA (gi: 9946855), S. pombe Obr1 (gi: 2462689), Sulfolobus solfataricus WrbA (gi: 15899861), Sulfolobus todakii WrbA (gi: 15621890), T. versicolor QR2 (gi: 12484052), Xylella fastidiosa WrbA (gi: 22993897), Yarrowia lipolytica Hyp (gi: 49647887), and Yersinia pestis WrbA (gi: 22126332).

FIG. 2.

Phylogenetic tree of selected WrbA, NAD(P)H:quinone oxidoreductase, and flavodoxin sequences. The full alignment of these sequences is shown in Fig. 1. The scale represents the average number of amino acid substitutions per site. WrbA_{E. coli} and WrbA_A. _fulgidus are in bold type. Organisms and ORF designations from the corresponding genomic sequence: A. fulgidus WrbA (gi: 11497955), A. thaliana FQR1 (gi: 21539481), B. cepacia WrbA (gi: 46310790), C. aurantiacus WrbA (gi: 53798190), C. crescentus WrbA (gi: 13422034), C. beijerinckii Fldx (gi: 1941945), D. gigas Fldx (gi: 40801), D. radiodurans WrbA (gi: 58177611), Exiguobacterium sp. strain 255-15 WrbA (gi: 68054352), E. carotovora WrbA (gi: 49613050), E. coli WrbA (gi: 148264) and Fldx (gi: 145986), G. trabeum QR1 (gi: 30027749) and QR2 (gi: 33668045), M. acetivorans WrbA (gi: 19915027), M. avium WrbA (gi: 41409133), M. barkeri WrbA (gi: 31793946), Mesorhizobium sp. strain BNC1 WrbA (gi: 68191386), M. degradans WrbA (gi: 48860599), M. mazei WrbA (gi: 21228326), N. crassa WrbA (gi: 38566940), N. punctiforme WrbA (gi: 23129682), P. arcticum WrbA (gi: 71038102), P. chrysosporium QR (gi: 4454993), Polaromonas sp. strain JS666 WrbA (gi: 67847813), P. aeruginosa WrbA (gi: 9946855), S. pombe Obr1 (gi: 2462689), S. solfataricus WrbA (gi: 15899861), S. todakii WrbA (gi: 15621890), T. versicolor QR2 (gi: 12484052), X. fastidiosa WrbA (gi: 22993897), Y. lipolytica WrbA (gi: 49647887), and Y. pestis WrbA (gi: 22126332).

FIG. 3.

Sequence alignment with WrbA sequences, Isf sequences, and misannotated WrbA sequences that have a compact cysteine motif. Numerical values at the C terminus indicate percent identity/percent similarity to E. coli WrbA. ns, not significant; #, completely conserved residues; = = = = = =, region of the flavodoxin signature motif; ▴, compact cysteine motif. Organisms and ORF designations from the corresponding genomic sequence: A. fulgidus Isf (gi: 11499480), A. fulgidus WrbA (gi: 11497955), Clostridium acetotrophicum WrbA (gi: 15896732), E. coli WrbA (gi: 148264), C. beijerinckii Fldx (gi: 1941945), Methanosarcina jannaschii Isf (gi: 15669271), Methanopyrus kandleri WrbA (gi: 20094374), and M. thermophila Isf (gi: 2246438).

FIG. 4.

UV-visible light spectra of reconstituted, oxidized WrbA_{E. coli} and WrbA_A. _fulgidus. Comparison of WrbA-bound FMN versus free FMN. WrbA_A. _fulgidus, —; WrbA_{E. coli}, —; free FMN, - - -.

FIG. 5.

pH dependence of apparent initial velocities. Activities were obtained at 37°C with WrbA_{E. coli} (○) and at 65°C with WrbA_A. _fulgidus (▴).

FIG. 6.

Arrhenius plot of apparent initial velocities. Activities were obtained at pH 7.2. WrbA_A. _fulgidus, ▴; WrbA_{E. coli}, ○.