title: KefF, the regulatory subunit of the potassium efflux system KefC, shows quinone oxidoreductase activity.
abstract: Escherichia coli and many other Gram-negative pathogenic bacteria protect themselves from the toxic effects of electrophilic compounds by using a potassium efflux system (Kef). Potassium efflux is coupled to the influx of protons, which lowers the internal pH and results in immediate protection. The activity of the Kef system is subject to complex regulation by glutathione and its S conjugates. Full activation of KefC requires a soluble ancillary protein, KefF. This protein has structural similarities to oxidoreductases, including human quinone reductases 1 and 2. Here, we show that KefF has enzymatic activity as an oxidoreductase, in addition to its role as the KefC activator. It accepts NADH and NADPH as electron donors and quinones and ferricyanide (in addition to other compounds) as acceptors. However, typical electrophilic activators of the Kef system, e.g., N-ethyl maleimide, are not substrates. If the enzymatic activity is disrupted by site-directed mutagenesis while retaining structural integrity, KefF is still able to activate the Kef system, showing that the role as an activator is independent of the enzyme activity. Potassium efflux assays show that electrophilic quinones are able to activate the Kef system by forming S conjugates with glutathione. Therefore, it appears that the enzymatic activity of KefF diminishes the redox toxicity of quinones, in parallel with the protection afforded by activation of the Kef system.
authors: School of Medical Sciences, University of Aberdeen, Aberdeen, AB25 2ZD, United Kingdom.
authors: Lisbeth
authors: L
authors: Lyngberg
authors: Jessica
authors: J
authors: Healy
authors: Wendy
authors: W
authors: Bartlett
authors: Samantha
authors: S
authors: Miller
authors: Stuart J
authors: SJ
authors: Conway
authors: Ian R
authors: IR
authors: Booth
authors: Tim
authors: T
authors: Rasmussen
mesh_terms: D016227:Benzoquinones / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005292:Ferricyanides / Q000378:metabolism; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000235:genetics / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D011188:Potassium / Q000378:metabolism; D015221:Potassium Channels / Q000378:metabolism; D021122:Protein Subunits / Q000378:metabolism
chemical_list: D016227:Benzoquinones; D029968:Escherichia coli Proteins; D005292:Ferricyanides; D015221:Potassium Channels; D021122:Protein Subunits; D009243:NAD; C007931:hexacyanoferrate III; C069287:KefC protein, E coli; C004532:quinone; D009249:NADP; C561939:KefF protein, E coli; D010088:Oxidoreductases; D016660:NAD(P)H Dehydrogenase (Quinone); D011188:Potassium