Bifidobacterium longum requires a fructokinase (Frk; ATP:D-fructose 6-phosphotransferase, EC 2.7.1.4) for fructose catabolism.
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 Co(2+)-based column, and the pH and temperature optima were determined. A biochemical analysis revealed that Frk displays the same affinity for fructose and ATP (Km(fructose) = 0.739 +/- 0.18 mM and Km(ATP) = 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.
Caescu
Cristina I
CI
Unité de Glycobiologie Structurale et Fonctionnelle, UMR CNRS-USTL 8576, Université des Sciences et Technologies de Lille, Villeneuve d'Ascq, France.
Vidal
Olivier
O
Krzewinski
Frédéric
F
Artenie
Vlad
V
Bouquelet
Stéphane
S
D001644:Bifidobacterium / Q000378:metabolism*; D002384:Catalysis; D005631:Fructokinases / Q000235:genetics / Q000502:physiology*; D005632:Fructose / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D014158:Transcription, Genetic
D011994:Recombinant Proteins; D005632:Fructose; D005631:Fructokinases; C046173:fructokinase; D005947:Glucose