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

Abstract

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 (K m fructose ‫؍‬ 0.739 ؎ 0.18 mM and K m 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.

Kinetic Analysis of Bifidobacterial Metabolism Reveals a Minor Role for Succinic Acid in the Regeneration of NAD + through Its Growth-Associated Production

“…4). Several factors related to the specific sugar consumption rate, such as the sugar uptake systems and the conversions necessary to incorporate a specific sugar into the fructose-6-phosphate pathway, can have great effects on which metabolic route is used (3,17). It has also been reported that in L. lactis the NADH ϩ H ϩ /NAD ϩ ratio (11) or the pool of ADP and ATP (30) can have an effect on changes in end product formation.…”

A Proteome Reference Map and Proteomic Analysis of Bifidobacterium longum NCC2705

“…Gene Expression Analysis by Semiquantitative RT-PCR-Under different growth conditions the relative abundance of mRNA of interest was determined by semiquantitative RT-PCR (10). Total RNAs was extracted from B. longum NCC2705 culture by using a MasterPure RNA purification kit (Epicenter Technologies, Madison, WI).…”

Lactose-over-Glucose Preference inBifidobacterium longumNCC2705:glcP, Encoding a Glucose Transporter, Is Subject to Lactose Repression

“…A sucrose permease gene from Bifidobacterium lactis was found as part of an operon that is induced by sucrose and raffinose and is subject to glucose repression (23). The isolation of a fructose kinase gene, frk, which is also repressed by glucose, has been related to fructose utilization in B. longum (3).…”