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Flavodoxin:Quinone Reductase (FqrB): a Redox Partner of Pyruvate:Ferredoxin Oxidoreductase That Reversibly Couples Pyruvate Oxidation to NADPH Production in Helicobacter pylori and Campylobacter jejuni

Martin St. Maurice ,^3,, Nunilo Cremades,^5,6, Matthew A. Croxen,^1,2,3 Gary Sisson,³ Javier Sancho,^5,6 and Paul S. Hoffman^1,2,3,4*

Department of Medicine, Division of Infectious Diseases and International Health,¹ Department of Microbiology, University of Virginia, Charlottesville, Virginia 22908,² Department of Microbiology and Immunology,³ Department of Medicine, Division of Infectious Diseases, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7,⁴ Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza,⁵ Biocomputing and Physics of Complex Systems Institute, Zaragoza Spain⁶

Received 23 February 2007/ Accepted 19 April 2007

Pyruvate-dependent reduction of NADP has been demonstrated in cell extracts of the human gastric pathogen Helicobacter pylori. However, NADP is not a substrate of purified pyruvate:ferredoxin oxidoreductase (PFOR), suggesting that other redox active enzymes mediate this reaction. Here we show that fqrB (HP1164), which is essential and highly conserved among the epsilonproteobacteria, exhibits NADPH oxidoreductase activity. FqrB was purified by nickel interaction chromatography following overexpression in Escherichia coli. The protein contained flavin adenine dinucleotide and exhibited NADPH quinone reductase activity with menadione or benzoquinone and weak activity with cytochrome c, molecular oxygen, and 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB). FqrB exhibited a ping-pong catalytic mechanism, a k_cat of 122 s^–1, and an apparent K_m of 14 µM for menadione and 26 µM for NADPH. FqrB also reduced flavodoxin (FldA), the electron carrier of PFOR. In coupled enzyme assays with purified PFOR and FldA, FqrB reduced NADP in a pyruvate- and reduced coenzyme A (CoA)-dependent manner. Moreover, in the presence of NADPH, CO₂, and acetyl-CoA, the PFOR:FldA:FqrB complex generated pyruvate via CO₂ fixation. PFOR was the rate-limiting enzyme in the complex, and nitazoxanide, a specific inhibitor of PFOR of H. pylori and Campylobacter jejuni, also inhibited NADP reduction in cell-free lysates. These capnophilic (CO₂-requiring) organisms contain gaps in pathways of central metabolism that would benefit substantially from pyruvate formation via CO₂ fixation. Thus, FqrB provides a novel function in pyruvate metabolism and, together with production of superoxide anions via quinone reduction under high oxygen tensions, contributes to the unique microaerobic lifestyle that defines the epsilonproteobacterial group.

Published ahead of print on 27 April 2007.

Present address: Department of Biochemistry, University of Wisconsin, Madison, WI 53706.

Martin St. Maurice and Nunilo Cremades contributed equally to this work and share first authorship.

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