Purification and properties of the mannokinase from Escherichia coli

Sebastián, Jesús; Asensio, Carlos

doi:10.1016/0003-9861(72)90492-4

Cited by 37 publications

(23 citation statements)

References 11 publications

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“…Bacterial fructokinases are usually mannofructokinases (45,49,52,53,54,65). Indeed, these enzymes convert both Dfructose and D-mannose to F6P and mannose-6-P, respectively.…”

Section: Discussionmentioning

confidence: 99%

“…Two procedures were used to measure fructokinase activity. Method I was the spectrometric method of Sebastian and Asensio (45), which couples the formation of F6P to the reduction of NADP ϩ via glucose phosphate isomerase (PGI; EC 5.3.1.9) and glucose-6-phosphate dehydrogenase. The standard assay mixture (200 l at 50°C) contained 100 mM sodium phosphate, 2.7 mM fructose, 10 mM ATP, and 10 mM MgCl 2 (pH 6.5).…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2004

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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.

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“…Bacterial fructokinases are usually mannofructokinases (45,49,52,53,54,65). Indeed, these enzymes convert both Dfructose and D-mannose to F6P and mannose-6-P, respectively.…”

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

et al. 2004

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“…We have shown (3) that a mutant of E. coli (HK 1691) in which the normal PTS-linked modes of fructose utilization were abolished gave rise to a further mutant (HK 1787) that grew on fructose by a novel route, in which fructose entered the cells by facilitated diffusion, followed by its phosphorylation to fructose 6-phosphate in an ATP-linked reaction catalyzed by a highly active manno(fructo)kinase (4). We also showed (3) that the facilitated diffusion of fructose was effected by an isoform (PtsG-F) of the principal glucose transporter, the normally PTS-linked protein PtsG, in which a valine had been replaced by a phenylalanine (V12F), the gene specified by ptsG maps at min 24.9 on the E. coli linkage map (5).…”

Section: Location Of the Gene Specifying Mak Activity On The E Coli mentioning

confidence: 99%

“…However, further mutants of S. typhimurium (2) and E. coli (3) have been described that grow on fructose even though they are unable to use other PT sugars. In the latter organisms, fructose has been shown (3) to enter the cells by facilitated diffusion by means of an isoform of the membrane-spanning protein PtsG, which is normally the primary port for the PT-dependent entry of glucose; the internalized ketose subsequently is phosphorylated to fructose 6-phosphate by ATP and a fructo(manno)kinase (4).…”

mentioning

confidence: 99%

Genetic control of manno(fructo)kinase activity in Escherichia coli

Sproul

Lambourne²,

D³

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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Mutants of Escherichia coli unable to use fructose by means of the phosphoenolpyruvate͞glycose phosphotransferase system mutate further to permit growth on that ketose by derepression of a manno(fructo)kinase (Mak ؉ phenotype) present in only trace amounts in the parent organisms (Mak-o phenotype). The mak gene was located at min 8.8 on the E. coli linkage map as an ORF designated yajF, of hitherto unknown function; it specifies a deduced polypeptide of 344 aa. The derepression of Mak activity was associated with a single base change at position 71 (codon 24) of the gene, where GCC (alanine) in Mak-o has been changed to GAC (aspartate) in Mak ؉ . By cloning selected portions of the total 1,032-bp mak gene into a plasmid that also carried a temperaturesensitive promoter, we showed that the mutation resided in a 117-bp region that does not specify sequences necessary for Mak activity but was located 46 bp upstream of a 915-bp portion that does. Mak ؉ and Mak-o strains differ greatly in the heat stability of the enzyme: at 61°C, mak-o cloned into a mak-o recipient loses 50% of its activity in approximately 6 min, whereas it takes over 30 min to achieve a similar reduction in the activity of mak ؉ cloned into a mak-o strain. However, the Mak activity of the cloned fragment specifying the enzyme without the regulatory region lost activity with a half-life of 29 min irrespective of whether it was derived from a mak ؉ or a mak-o donor, which indicates that the A24D mutation contributes to the high enzyme activity of Mak ؉ mutants by serving to protect Mak from denaturation.

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“…one unit of MDH activity was defined as the amount of enzyme catalyzing the reduction of 1 µmol of D-fructose per minute under the above assay conditions. Fructokinase (FK) activity was assayed according to spectrophotometric method of Sebastian and Asensio (42). the standard assay mixture (200 µl at 30 °c) contained 100 mmol/l sodium phosphate, 2.7 mmol/l fructose, 10 mol/l AtP, and 10 mmol/l Mgcl 2 (ph 6.5).…”

Section: Methodsmentioning

confidence: 99%