In vitro reconstitution of an NADPH-dependent superoxide reduction pathway from Pyrococcus furiosus
Document Type
Article
Publication Date
2005
Abstract
A scheme for the detoxification of superoxide in Pyrococcus furiosus has been previously proposed in which superoxide reductase (SOR) reduces (rather than dismutates) Superoxide to hydrogen peroxide by using electrons from reduced rubredoxin (Rd). Rd is reduced with electrons from NAD(P)H by the enzyme NAD(P)H: rubredoxin oxidoreductase (NROR). The goal of the present work was to reconstitute this pathway in vitro using recombinant enzymes. While recombinant forms of SOR and Rd are available, the gene encoding P. furiosus NROR (PF1197) was found to be exceedingly toxic to Escherichia coli, and an active recombinant form (rNROR) was obtained via a fusion protein expression system, which produced an inactive form of NROR until cleavage. This allowed the complete pathway from NAD(P)H to the reduction of SOR via NROR and Rd to be reconstituted in vitro using recombinant proteins. rNROR is a 39.9-kDa protein whose sequence contains both flavin adenine dinucleotide (FAD)- and NAD(P)H-binding motifs, and it shares significant similarity with known and putative Rd-dependent oxidoreductases from several anaerobic bacteria, both mesophilic and hyperthermophilic. FAD was shown to be essential for activity in reconstitution assays and could not be replaced by flavin mononucleotide (FMN). The bound FAD has a midpoint potential of -173 mV at 23°C (-193 mV at 80°C). Like native NROR, the recombinant enzyme catalyzed the NADPH-dependent reduction of rubredoxin both at high (80°C) and low (23°C) temperatures, consistent with its proposed role in the superoxide reduction pathway. This is the first demonstration of in vitro superoxide reduction to hydrogen peroxide using NAD(P)H as the electron donor in an SOR-mediated pathway. Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Publication Title
Applied and Environmental Microbiology
Volume
71
Issue
3
First Page
1522
Last Page
1530
Recommended Citation
Grunden, A. M.; Jenney, Francis E. Jr.; Ma, K.; Ji, M.; Weinberg, M. V.; and Adams, M. W., "In vitro reconstitution of an NADPH-dependent superoxide reduction pathway from Pyrococcus furiosus" (2005). PCOM Scholarly Papers. 582.
https://digitalcommons.pcom.edu/scholarly_papers/582
Comments
This article was published in Applied and Environmental Microbiology, Volume 71, Issue 3, Pages 1522-1530.
The published version is available at http://dx.doi.org/10.1128/AEM.71.3.1522-1530.2005.Copyright © 2005 ASM.