Construction of a new catabolic pathway for d-fructose in Escherichia coli K12 using an l-sorbose-specific enzyme from Klebsiella pneumoniae (original) (raw)
Related papers
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.
Carbohydrate Research, 2001
Not only sucrose but the five isomeric a-D-glucosyl-D-fructoses trehalulose, turanose, maltulose, leucrose, and palatinose are utilized by Klebsiella pneumoniae as energy sources for growth, thereby undergoing phosphorylation by a phosphoenolpyruvate-dependent phosphotransferase system uniformly at O-6 of the glucosyl moiety. Similarly, maltose, isomaltose, and maltitol, when exposed to these conditions, are phosphorylated regiospecifically at O-6 of their non-reducing glucose portion. The structures of these novel compounds have been established unequivocally by enzymatic analysis, acid hydrolysis, FAB negative-ion spectrometry, and 1 H and 13 C NMR spectroscopy. In cells of K. pneumoniae, hydrolysis of sucrose 6-phosphate is catalyzed by sucrose 6-phosphate hydrolase from Family 32 of the glycosylhydrolase superfamily. The five 6%-O-phosphorylated a-D-glucosyl-fructoses are hydrolyzed by an inducible ( 49-50 Kda) phospho-a-glucosidase from Family 4 of the glycosylhydrolase superfamily.
European Journal of Biochemistry, 1979
A phosphoryl exchange reaction between fructose 1-phosphate and fructose was found to be catalyzed by a membrane preparation isolated from Bacillus subtilis. The regulation of the biosynthesis of the activity in the wild type as well as in the regulation mutants fruB closely correlates with that of the membrane-bound enzyme I1 of the phosphoenolpyruvate fructose l-phosphotransferase system which is known to mediate the transmembrane vectorial phosphorylation of fructose. The computed analysis of the kinetic data shows that the mechanism of the enzyme I1 is pingpong, i.e. that a phosphoryl-enzyme intermediate occurs in the reaction. The apparent dissociation constants of the enzyme II/fructose 1-phosphate complex and of the phosphoryl enzyme II/fructose complex are estimated. The value of the standard free energy of the hydrolysis of the bond between the phosphoryl moiety and the enzyme suggests a covalent bonding. This intermediate is assumed to occur in the physiological functioning of the enzyme which utilizes the phosphocarrier protein HPr as phosphoryl donor. The exchange reaction is competitively inhibited by high fructose concentrations : this indicates that the same site of the enzyme binds fructose and fructose 1-phosphate, this site being accessible to fructose on the external side of the membrane when the enzyme is phosphorylated. Since the discovery by Kundig et al. [l] of bacterial phosphotransferase systems, it has been shown that these systems mediate the first step of the catabolism of several carbohydrates in many bacterial species (see recent reviews by Postma and Roseman [2], and Hengstenberg [ 3 ]). Genetic evidence was presented that in Bacillus subtilis fructose, glucose, mannose, mannitol and sucrose phosphorylation was dependent upon such systems [4]. A detailed study of the catabolism of either intracellular or extracellular fructose [5-71 allowed us to demonstrate that an inducible phosphoenolpyruvate fructose 1-phosphotransferase system was devoted to a physiological vectorial phosphorylation step, i.e. an inwards translocation of the sugar concomitant with the phosphorylation at the C-1 site. The system involves at least three components: enzyme I, the phosphocarrier protein HPr, and a membrane-bound enzyme 11; the overall reaction may be schematized in its simplest form, as follows: Abbreviation. HPr protein, heat-stable phosphocarrier protein. Enzyme. Phosphoenolpyruvate-fructose phosphotransferase (EC 2.7.1.98).
Fructose bisphosphatase from Escherichia coli. Purification and characterization
Archives of Biochemistry and Biophysics, 1983
Escherichia coli fructose-l,&bisphosphatase has been purified for the first time, using a clone containing an approximately 50-fold increased amount of the enzyme. The procedure includes chromatography in phosphocellulose followed by substrate elution and gel filtration. The enzyme has a subunit molecular weight of approximately 40,000 and in nondenaturing conditions is present in several aggregated forms in which the tetramer seems to predominate at low enzyme concentrations. Fructose bisphosphatase activity is specific for fructose 1,6-bisphosphate (K, of approximately 5 PM), shows inhibition by substrate above 0.05 mM, requires Me for catalysis, and has a maximum of activity around pH 7.5. The enzyme is susceptible to strong inhibition by AMP (50% inhibition around 15 PM). Phosphoenolpyruvate is a moderate inhibitor but was able to block the inhibition by AMP and may play an important role in the regulation of fructose bisphosphatase activity in wivo. Fructose 2,6-bisphosphate did not affect the rate of reaction. Fructose bisphosphatase (EC 3.1.3.11, D-fructose-1,6-bisphosphate l-phosphohydrolase) catalyzes an essential reaction of the gluconeogenic pathway and plays an important role in its regulation (1). The enzyme from several sources has been purified and characterized, but it is mainly the enzyme from mammalian tissues that has been extensively studied and whose structure and mechanism of action are relatively well known (2). The physiological role of the enzyme in Escherichia coli was shown through the use of mutants (3, 4), but the last report about the enzyme from
Journal of Bacteriology, 2000
In Escherichia coli, gene products of the glp regulon mediate utilization of glycerol and sn-glycerol 3-phosphate. The glpFKX operon encodes glycerol diffusion facilitator, glycerol kinase, and as shown here, a fructose 1,6-bisphosphatase that is distinct from the previously described fbp-encoded enzyme. The purified enzyme was dimeric, dependent on Mn 2؉ for activity, and exhibited an apparent K m of 35 M for fructose 1,6-bisphosphate. The enzyme was inhibited by ADP and phosphate and activated by phosphoenolpyruvate.
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.
Biochemistry, 1997
A bifunctional enzyme, fructose-6-phosphate 2-kinase-fructose 2,6-bisphosphatase, catalyzes synthesis and degradation of fructose 2,6-bisphosphate. Mutants of basic residues, including Lys51, Arg78, Arg79, Arg136, Lys172, and Arg193, immediately around the active site of rat testis fructose 6-P,2kinase were constructed, and their steady state kinetics, ATP binding, and the effect of pH on the kinetics were characterized. All mutants showed a several-fold increase in K MgATP , much larger increases in K Fru 6-P , and decreased V compared to those of the wild type enzyme (WT). Replacement of Lys172 and Arg193 with Ala and Leu, respectively, also produced mutants with large K Fru 6-P values. Substitution of Lys51, which is located in a Walker-A motif (GXXGXGKT, amino acids 45-52), with Ala or His resulted in enzymes with increased K MgATP values and unable to bind Fru 6-P. The dissociation constants for 2′-(3′)-O-(N-methylanthraniloyl)-ATP (mantATP) and ATP of all these mutants except Lys51 were similar. Lys51 mutants were unable to bind mantATP. The pH dependence of V and the V/Ks for MgATP and Fru 6-P suggest a mechanism in which reactants and enzyme combine irrespective of the protonation state of groups required for binding and catalysis, but only the correctly protonated enzyme-substrate complex is catalytically active. A chemical mechanism is suggested in which a general base accepts a proton from the 2-hydroxyl of Fru 6-P concomitant with nucleophilic attack on the γ-phosphate of MgATP. Phosphoryl transfer is also facilitated by interaction of the γ-phosphate with a positively charged residue that neutralizes the remaining negative charge. The dianionic form of the 6-phosphate of fructose 6-P is required for binding, and it is likely anchored by a positively charged enzyme residue. A comparison of the pH dependence of kinetic parameters for Ala or His mutant proteins at Lys51, Lys172, and Arg79 suggests that Lys51 interacts with the γ-phosphate of MgATP and that several other arginines likely participate in transition state stabilization of the transferred phosphoryl. The active site general base has yet to be identified.
Frontiers in Bioengineering and Biotechnology, 2021
Fructose utilization in Corynebacterium glutamicum starts with its uptake and concomitant phosphorylation via the phosphotransferase system (PTS) to yield intracellular fructose 1-phosphate, which enters glycolysis upon ATP-dependent phosphorylation to fructose 1,6-bisphosphate by 1-phosphofructokinase. This is known to result in a significantly reduced oxidative pentose phosphate pathway (oxPPP) flux on fructose (∼10%) compared to glucose (∼60%). Consequently, the biosynthesis of NADPH demanding products, e.g., L-lysine, by C. glutamicum is largely decreased when fructose is the only carbon source. Previous works reported that fructose is partially utilized via the glucose-specific PTS presumably generating fructose 6phosphate. This closer proximity to the entry point of the oxPPP might increase oxPPP flux and, consequently, NADPH availability. Here, we generated deletion strains lacking either the fructose-specific PTS or 1-phosphofructokinase activity. We used these strains in short-term evolution experiments on fructose minimal medium and isolated mutant strains, which regained the ability of fast growth on fructose as a sole carbon source. In these fructose mutants, the deletion of the glucose-specific PTS as well as the 6-phosphofructokinase gene, abolished growth, unequivocally showing fructose phosphorylation via glucose-specific PTS to fructose 6-phosphate. Gene sequencing revealed three independent amino acid substitutions in PtsG (M260V, M260T, and P318S). These three PtsG variants mediated faster fructose uptake and utilization compared to native PtsG. In-depth analysis of the effects of fructose utilization via these PtsG variants revealed significantly increased ODs, reduced side-product accumulation, and increased L-lysine production by 50%.
Microbial Cell Factories
Background: The halophilic bacterium Chromohalobacter salexigens metabolizes glucose exclusively through the Entner-Doudoroff (ED) pathway, an adaptation which results in inefficient growth, with significant carbon overflow, especially at low salinity. Preliminary analysis of C. salexigens genome suggests that fructose metabolism could proceed through the Entner-Doudoroff and Embden-Meyerhof-Parnas (EMP) pathways. In order to thrive at high salinity, this bacterium relies on the biosynthesis and accumulation of ectoines as major compatible solutes. This metabolic pathway imposes a high metabolic burden due to the consumption of a relevant proportion of cellular resources, including both energy molecules (NADPH and ATP) and carbon building blocks. Therefore, the existence of more than one glycolytic pathway with different stoichiometries may be an advantage for C. salexigens. The aim of this work is to experimentally characterize the metabolism of fructose in C. salexigens. Results: Fructose metabolism was analyzed using in silico genome analysis, RT-PCR, isotopic labeling, and genetic approaches. During growth on fructose as the sole carbon source, carbon overflow was not observed in a wide range of salt concentrations, and higher biomass yields were reached. We unveiled the initial steps of the two pathways for fructose incorporation and their links to central metabolism. While glucose is metabolized exclusively through the Entner-Doudoroff (ED) pathway, fructose is also partially metabolized by the Embden-Meyerhof-Parnas (EMP) route. Tracking isotopic label from [1-13 C] fructose to ectoines revealed that 81% and 19% of the fructose were metabolized through ED and EMP-like routes, respectively. Activities of enzymes from both routes were demonstrated in vitro by 31 P-NMR. Genes encoding predicted fructokinase and 1-phosphofructokinase were cloned and the activities of their protein products were confirmed. Importantly, the protein encoded by csal1534 gene functions as fructose bisphosphatase, although it had been annotated previously as pyrophosphate-dependent phosphofructokinase. The gluconeogenic rather than glycolytic role of this enzyme in vivo is in agreement with the lack of 6-phosphofructokinase activity previously described.