Abstract
We recently demonstrated the presence of a quinone detoxification pathway present in Firmicutes. It is based on two enzyme activities, namely a quinone reductase, YaiB, described here, and a hydroquinone dioxygenase, YaiA, described in a separate protocol. In Lactococcus lactis (L. lactis), these enzymes are encoded by the yahCD-yaiAB operon. The operon is induced by copper to prevent the synergistic toxicity of quinones and copper. The quinone reductase, YaiB, reduces p-benzoquinone and a range of quinone derivatives to hydroquinone, using NADPH as a reductant, according to the reaction: p-benzoquinone + NADPH + H+ → hydroquinone + NADP+. We here describe the measurement of quinone reductase activity, based on the spectrophotometric measurement of NADPH-oxidation.
Materials and Reagents
Equipment
Procedure
Representative data
Figure 1. Representative data. Hanes-Woolf plot of p-benzoquinone reduction by YaiB of L. lactis. [S] is the mM substrate concentration and v the reaction rate in mmol/min. The slope of the linear regression line equals 1/vmax: vmax = 1/0.474 = 2.11 mmol/min/mg. Km is defined by the intercept of the regression line with the ordinate, which equals Km/vmax: Km = 0.1338*2.11 = 0.28 mM.
Notes
Recipes
Acknowledgments
This work was supported by Russian Federation Government Grant 14.Z50.31.0011 to leading scientists. The procedure has previously been described in Mancini et al. (2015).
References
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