Abstract
We recently demonstrated the presence of a quinone detoxification pathway present in Firmicutes. It is based on two enzyme activities, namely a hydroquinone dioxygenase, YaiA, described here, and a hydroquinone reductase, YaiB, 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 hydroquinone dioxygenase, YaiA, converts hydroquinones to 4-hydroxymuconic semialdehyde, using molecular oxygen as oxidant according to the reaction: hydroquinone + O2 → 4-hydroxymuconic semialdehyde + H+ We here describe two methods for measurements for hydroquinone dioxygenase activity, based on oxygen consumption measured with an oxygen electrode and the spectrophotometric detection of 4-hydroxymuconic semialdehyde. Both assays are conducted with crude cell extracts.
Materials and Reagents
Equipment
Procedure
Representative data
Figure 1. Absorption spectra of hydroquinone conversion to 4-hydroxymuconic semialdehyde. The lower curve was recorded at time 0 and the upper curve after 60 min of reaction. Inset, difference spectrum recorded between YaiA-containing extract incubated with and without 1 mM hydroquinone for 1 h, showing the absorption maximum of the reaction product at 320 nm. Axis labels are the same as in the main figure. Note that the spectra will look differently if substituted hydroquinones are used as the substrate for YaiA, but are not expected to change for different YaiA-like enzymes. Figure 2. Oxygen consumption by hydroquinone dioxygenase activity in crude extracts. The reactions were induced by the addition of 1 mM hydroquinone at 2.5 min. Curves from top to bottom are: 1 mg of cytoplasmic extract from E. coli not expressing hydroquinone dioxygenase, extract heated to 95 °C for 5 min, and 1 mg of cytoplasmic extract from E. coli strain expressing YaiA of Lactococcus lactis from a plasmid.
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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