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Journal of Bacteriology, October 2004, p. 6515-6525, Vol. 186, No. 19
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.19.6515-6525.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Bifidobacterium longum Requires a Fructokinase (Frk; ATP:D-Fructose 6-Phosphotransferase, EC 2.7.1.4) for Fructose Catabolism

Cristina I. Caescu,1,2 Olivier Vidal,1* Frédéric Krzewinski,1 Vlad Artenie,2 and Stéphane Bouquelet1

Unité de Glycobiologie Structurale et Fonctionnelle, UMR CNRS-USTL 8576, Université des Sciences et Technologies de Lille, Villeneuve d'Ascq, France,1 Laboratorul de Biochimie, Universitatea "Al. I. Cuza," Iasi, Romania2

Received 23 February 2004/ Accepted 28 June 2004

Although the ability of Bifidobacterium spp. to grow on fructose as a unique carbon source has been demonstrated, the enzyme(s) needed to incorporate fructose into a catabolic pathway has hitherto not been defined. This work demonstrates that intracellular fructose is metabolized via the fructose-6-P phosphoketolase pathway and suggests that a fructokinase (Frk; EC 2.7.1.4) is the enzyme that is necessary and sufficient for the assimilation of fructose into this catabolic route in Bifidobacterium longum. The B. longum A10C fructokinase-encoding gene (frk) was expressed in Escherichia coli from a pET28 vector with an attached N-terminal histidine tag. The expressed enzyme was purified by affinity chromatography on a Co2+-based column, and the pH and temperature optima were determined. A biochemical analysis revealed that Frk displays the same affinity for fructose and ATP (Kmfructose = 0.739 ± 0.18 mM and KmATP = 0.756 ± 0.08 mM), is highly specific for D-fructose, and is inhibited by an excess of ATP (>12 mM). It was also found that frk is inducible by fructose and is subject to glucose-mediated repression. Consequently, this work presents the first characterization at the molecular and biochemical level of a fructokinase from a gram-positive bacterium that is highly specific for D-fructose.

* Corresponding author. Mailing address: Unité de Glycobiologie Structurale et Fonctionnelle, UMR CNRS-USTL 8576, Université des Sciences et Technologies de Lille, F-59655 Villeneuve d'Ascq, France. Phone: (33) 3 20 43 41 42. Fax: (33) 3 20 43 65 55. E-mail: olivier.vidal{at}univ-lille1.fr.

Journal of Bacteriology, October 2004, p. 6515-6525, Vol. 186, No. 19
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.19.6515-6525.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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