A Redox‐Based Superoxide Generation System Using Quinone/Quinone Reductase. (26th June 2018)

• Record Type: Journal Article
• Authors: Singh, Shailesh Kumar; Husain, Syed Masood
• Is Part Of: Chembiochem. Volume 19:Number 15(2018)
• Page Start: 1657
• Page End: 1663
• Publication Date: 2018-06-26
• Subjects: enzyme catalysis -- oxygen -- quinones -- radicals -- redox chemistry; Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pharmaceutical chemistry -- Periodicals; 572
• DOI: 10.1002/cbic.201800071
• Languages: English
• ISSNs: 1439-4227

Abstract

Abstract: Superoxide (O2 .− ) generation in biological systems is achieved through some of the most complex enzymatic systems. Of these, only xanthine/xanthine oxidase has been used for in vitro biochemical studies. However, it suffers from limitations such as a lack of suitable heterologous expression system for xanthine oxidase and the irreversible consumption and low solubility of xanthine under physiological conditions. Herein, we report a redox‐based, enzyme‐catalyzed system, in which autoxidation of hydroquinone to quinone via semiquinone results in superoxide generation. Quinone is reduced back to hydroquinone by using the NfsB (oxygen‐insensitive nitroreductase) enzyme of Escherichia coli strain K‐12 and nicotinamide adenine dinucleotide phosphate hydride (NADPH; which is regenerated by using the glucose/glucose dehydrogenase system). This new system relies on quinones that can be recycled and have superior water solubility, as well as enzymes that are heterologously expressed. By using a variety of quinones and reaction conditions, along with a comparison of real‐time fluorescence, menadione has been identified as the optimal substrate for superoxide generation. The new redox‐based system presents a viable alternative for studying the biochemistry of superoxide under different physiological and pathological conditions. Abstract : Creating superspecies : A redox‐based, enzyme‐catalyzed system for superoxide (O2 .− ) generation is achieved through autoxidation of hydroquinone via semiquinone. To convert quinone to hydroquinone, a quinone/quinone reductase system is used. The system not only recycles quinones, which have superior water solubility, but also uses enzymes that are heterologously expressed.