Alternate pathways of D-fructose transport and metabolism in (original) (raw)
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Pathways of d-fructose transport in Arthrobacter pyridinolis
Archives of Biochemistry and Biophysics, 1974
Previous work indicated that Arthrobacter pyridinolis can transport n-fructose by either a phosphoenolpyruvate:n-fructose phosphotransferase system or by a respiration-coupled system. The respiration-coupled transport system for n-fructose, which is stimulated by the addition of L-malate, has been characterized in membrane vesicles from n-fructose-grown cells. Such vesicles carry out malate-dependent uptake of n-fructose but not of n-glucose or n-rhamnose, indicating that there is a sugar-specific component to the respiration-coupled transport system. A mutant which is deficient in the n-fructose-specific component was isolated. Vesicles from fructose-glutamate-grown cells of a phosphotransferase-negative strain (APlOO) exhibited malate-dependent n-fructose uptake, while phosphoenolpyruvate-dependent uptake was reduced to a small fraction of that seen with vesicles from wild-type cells. Inhibitors of electron transport, carbonyl cyanide m-chlorophenyl hydra. zone, 2,4-dinitrophenol and N-ethylmaleimide caused marked inhibition of malatedependent D-frUCtOSe uptake while exerting little or no effect on p,hosphoenolpyruvate-dependent transport of the sugar in vesicles from wild-type cells. Activity of a flavin adenine dinucleotide-linked L-malic dehydrogenase was detected in membrane vesicles as well as in whole cells.
Microbiology, 1998
Aeromonas hydrophila was examined for fructose and mannose transport systems. A. hydrophila was shown to possess a phosphoenolpyruvate (PEP) : fructose phosphotransferase system (fructose-PTS) and a mannosespecific PTS, both induced by fructose and mannose. The mannose-PTS of A. hydrophila exhibited cross-reactivity with Escherichia coli mannose-PTS proteins. The fructose-PTS proteins exhibited cross-reactivities with E. coli and Xanthomonas campestris fructose-PTS proteins. In A. hydrophila grown on mannose as well as on fructose, the phosphorylated derivative accumulated from fructose was fructose 1-phosphate. Identification of fructose 1-phosphate was confirmed by 13 C-NMR spectroscopy. 1-Phosphofructokinase (1-PFK), which converts the product of the PTS reaction to fructose 1,6-diphosphate, was present in A. hydrophila grown with fructose but not on mannose. An inducible phosphofructomutase (PFM) activity, an unusual enzyme converting fructose 1-phosphate to fructose 6-phosphate, was detected in extracts induced by mannose or fructose. These results suggest that in cells grown on fructose, fructose 1-phosphate could be converted to fructose 1,6-diphosphate either directly by the 1-PFK activity or via fructose 6-phosphate by the PFM and 6-phosphofructokinase activities. In cells grown on mannose, the degradation of fructose 1-phosphate via PFM and the Embden-Meyerhof pathway appeared to be a unique route.
Journal of bacteriology, 1974
The accumulation of 5-keto-D-fructose (5KF) by Gluconobacter cerinus grown on D-fructose in unbuffered medium was shown to be optimal at pH 4.0 after cell growth ceased. During the exponential phase of growth or at neutral pH after the onset of the stationary phase, 5KF production continued but did not accumulate because of its rapid reutilization by reduction to D-fructose. The extent of isotope incorporation into C5 of ribonucleic acid ribose when cells were grown in the presence of specifically labeled D-glucose and D-fructose clearly indicated that (i) the hexose monophosphate oxidative pathway is the predominant metabolic route for carbohydrate assimilation and (ii) extensive randomization of label between Cl and C6 of D-fructose occurred prior to its conversion into pentose. It is suggested that the cyclic oxidation and reduction through the symmetrical 5KF molecule, which accounts for the observed randomization of isotope in D-fructose, provides the cells with an effective mechanism for the regeneration of nicotinamide adenine dinucleotide phosphate during the period of intensive growth.
Proceedings of the National Academy of Sciences, 2000
From mutants of Escherichia coli unable to utilize fructose via the phosphoenolpyruvate͞glycose phosphotransferase system (PTS), further mutants were selected that grow on fructose as the sole carbon source, albeit with relatively low affinity for that hexose (Km for growth Ϸ8 mM but with Vmax for generation time Ϸ1 h 10 min); the fructose thus taken into the cells is phosphorylated to fructose 6-phosphate by ATP and a cytosolic fructo(manno)kinase (Mak). The gene effecting the translocation of fructose was identified by Hfr-mediated conjugations and by phage-mediated transduction as specifying an isoform of the membrane-spanning enzyme II Glc of the PTS, which we designate ptsG-F. Exconjugants that had acquired ptsG ؉ from Hfr strains used for mapping (designated ptsG-I) grew very poorly on fructose (Vmax Ϸ7 h 20 min), even though they were rich in Mak activity. A mutant of E. coli also rich in Mak but unable to grow on glucose by virtue of transposonmediated inactivations both of ptsG and of the genes specifying enzyme II Man (manXYZ) was restored to growth on glucose by plasmids containing either ptsG-F or ptsG-I, but only the former restored growth on fructose. Sequence analysis showed that the difference between these two forms of ptsG, which was reflected also by differences in the rates at which they translocated mannose and glucose analogs such as methyl ␣-glucoside and 2-deoxyglucose, resided in a substitution of G in ptsG-I by T in ptsG-F in the first position of codon 12, with consequent replacement of valine by phenylalanine in the deduced amino acid sequence.
Biochimica et biophysica acta, 1973
Mutants of Arthrobacter pyridinolis which were deficient in phosphoenolpyruvate:fructose phosphotransferase activity were studied using in vitro complementation assays. In this way the strains were divided into those deficient in the inducible membrane-bound component and into three groups which were deficient in different soluble ccmponents. One of the soluble components was inducible. Phosphoenolpyruvate:ffuctose phosphotransferase activity was also demonstrated in membrane vesicles prepared from fructose-grown A. pyridinolis. The activity was correlated with transport of fructose into the vesicles. Some kinetic parameters and temFerature optima for the transport process in vesicles were studied.
…, 1996
A strain of Zygosscchammyces bailii was selected for studies on fructose and glucose transport to determine the basis of the fructophilic behaviour of this species. Fructose was transported by a specific low-aff inity, high-capacity transport system with a Km of 65.6 mM and a Vmu of 6 7 mmol g-l h-' for cells grown on 2% (w/v) fructose, while the transport of glucose showed a Km of 7 mM and a Vmu of 1.7 mmol g-1 h-l for cells grown on 2 % (w/v) glucose. The transporter of glucose also accepted fructose as a substrate. Fructose inactivated the glucose transporter; inactivation was faster at higher concentrations. Both transporters were partially inductive. Measurements of metabolic fluxes and respiration and fermentation rates supported the general features identified by transport measurements. The kinetics and regulation of transport of the two sugars confirm the fructophilic behaviour previously described by other authors.
The Journal of biological chemistry, 1970
A specific reduced nicotinamide dinucleotide phosphatelinked 5-keto-D-fructose reductase has been isolated and purified approximately 300-fold from extracts of bakers' yeast. The reaction catalyzed by the enzyme was found to be 5-Keto-D-fructose + NADPH + H+ + L-sorbose-l-NADP+ K, values for NADPH and 5keto-D-fructose were 4.6 x 10V5 M and 1 X 10e3 M, respectively. All attempts to demonstrate reversibility of the reaction were unsuccessful. Although L-sorbose, at exceedingly high concentrations, does not affect the rate of NADPH oxidation by 5-keto-D-fructose, NADP+ was found to be a competitive inhibitor with a Ki value of 1.3 X 10-4M. Of all other substrates examined, only 5-keto-D-fructose phosphate was active. The Km value for the phosphate ester was found to be 2.9 X lop4 M with a maximal rate of reduction of only 1.8% of that observed for 5-keto-D-fructose. It has been established that the yeast reductase as well as sheep liver NAD-specific sorbitol dehydrogenase and Candid4
Ffz1, a new transporter specific for fructose from Zygosaccharomyces bailii
Microbiology, 2004
The basis of fructophily in the yeast Zygosaccharomyces bailii has been shown to reside in the performance of transport systems for hexoses. In this study, a gene encoding a fructose-specific transporter was characterized. The strategy involved the functional complementation of a Saccharomyces cerevisiae strain that does not take up hexoses (hxt-null strain). This strain was transformed with a genomic library of Z. bailii. One transformant capable of growing on fructose, but not on glucose, was obtained. This transformant did not transport D-[ 14 C]glucose, and the kinetic parameters for D-[ 14 C]fructose were V max =3?3 mmol h "1 g "1 and K m =80?4 mM. As in the original strain of Z. bailii, fructose uptake was not inhibited by the presence of other hexoses or uranyl. The plasmid responsible for the observed phenotype was found to carry an ORF encoding a 616 amino acid protein with the characteristics of a membrane transporter, which was designated FFZ1 (fructose facilitator Zygosaccharomyces). The impairment in function observed in an S. cerevisiae transformant expressing a truncated Ffz1 protein lacking 67 amino acids at the C-terminus suggests an important role for this terminal part in the proper structure of the transporter.