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J. Bacteriol. doi:10.1128/JB.00287-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

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

Department of Medicine, Division of Infectious Diseases and International Health, Department of Microbiology, University of Virginia, Charlottesville, Va. 22908; Departments of Microbiology and Immunology and Medicine, Division of Infectious Diseases, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7; and Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, and Biocomputing and Physics of Complex Systems Institute (BIFI), Spain

^* To whom correspondence should be addressed. Email: psh2n{at}virginia.edu.

	Abstract

Pyruvate dependent reduction of NADP has been demonstrated in cell-free extracts of the human gastric pathogen Helicobacter pylori. However, NADP is not a substrate of purified pyruvate: ferredoxin oxidoreductase (PFOR), suggesting other redox active enzymes mediate this reaction. Here we show that fqrB (HP1164), which is essential and highly conserved among the epsilon proteobacteria, exhibits NADPH - oxidoreductase activity. FqrB was purified by nickel interaction chromatography following over expression in E. coli. The protein contained FAD and exhibited NADPH quinone reductase activity with menadione or benzoquinone and weak activity with cytochrome c, molecular oxygen and DTNB. FqrB exhibited a ping pong catalytic mechanism, a k_cat of 122s^-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 pyruvate: ferredoxin oxidoreductase (PFOR). In coupled enzyme assays with purified PFOR and FldA, FqrB reduced NADP in a pyruvate and reduced coenzyme A-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 C. 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 epsilon proteobacterial group.

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