Glucosamine-6-phosphate synthase from Escherichia coli: determination of the mechanism of inactivation by N3-fumaroyl-L-2,3-diaminopropionic derivatives
Abstract:A mechanistic investigation of the inactivation of Escherichia coli glucosamine-6-phosphate synthase by N3-(4-methoxyfumaroyl)-L-2,3-diaminopropionate (FMDP) was undertaken. On the basis of the known participation of the N-terminal cysteine residue in this process [Chmara et al. (1986) Biochim. Biophys. Acta 870, 357; Badet et al. (1988) Biochemistry 27, 2282], the model reactions between FMDP and L-cysteine and between FMDP and the synthetic decapeptide Cys-Gly-Ile-Val-Gly-Ala-Ile-Ala-Gln-Arg, corresponding t… Show more
“…Especially the occurrence of an additional NH-signal in the 15 N- 1 H-HSQC spectrum and a coupling of the OC(=O)NH proton to this NH in the TOCSY spectrum confirmed this reaction. Notably, such a transcyclization reaction between cysteines coupled to maleimides was already reported, but only in few examples in literature [34–36]. Scheme 3Schematic illustration of the irreversible transcyclization reaction of 4A leading to the secondary species 4B
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The high mortality rate of lung cancer patients and the frequent occurrence of side effects during cancer therapy demonstrate the need for more selective and targeted drugs. An important and well-established target for lung cancer treatment is the occasionally mutated epidermal growth factor receptor (EGFR). As platinum(II) drugs are still the most important therapeutics against lung cancer, we synthesized in this study the first platinum(IV) complexes coupled to the EGFR-targeting peptide LARLLT (and the shuffled RTALLL as reference). Notably, HPLC–MS measurements revealed two different peaks with the same molecular mass, which turned out to be a transcyclization reaction in the linker between maleimide and the coupled cysteine moiety. With regard to the EGFR specificity, subsequent biological investigations (3-day viability, 14-day clonogenic assays and platinum uptake) on four different cell lines with different verified EGFR expression levels were performed. Unexpectedly, the results showed neither an enhanced activity nor an EGFR expression-dependent uptake of our new compounds. Consequently, fluorophore-coupled peptides were synthesized to re-evaluate the targeting ability of LARLLT itself. However, also with these molecules, flow cytometry measurements showed no correlation of drug uptake with the EGFR expression levels. Taken together, we successfully synthesized the first platinum(IV) complexes coupled to an EGFR-targeting peptide; however, the biological investigations revealed that LARLLT is not an appropriate peptide for enhancing the specific uptake of small-molecule drugs into EGFR-overexpressing cancer cells.Electronic supplementary materialThe online version of this article (doi:10.1007/s00775-017-1450-7) contains supplementary material, which is available to authorized users.
“…Especially the occurrence of an additional NH-signal in the 15 N- 1 H-HSQC spectrum and a coupling of the OC(=O)NH proton to this NH in the TOCSY spectrum confirmed this reaction. Notably, such a transcyclization reaction between cysteines coupled to maleimides was already reported, but only in few examples in literature [34–36]. Scheme 3Schematic illustration of the irreversible transcyclization reaction of 4A leading to the secondary species 4B
…”
The high mortality rate of lung cancer patients and the frequent occurrence of side effects during cancer therapy demonstrate the need for more selective and targeted drugs. An important and well-established target for lung cancer treatment is the occasionally mutated epidermal growth factor receptor (EGFR). As platinum(II) drugs are still the most important therapeutics against lung cancer, we synthesized in this study the first platinum(IV) complexes coupled to the EGFR-targeting peptide LARLLT (and the shuffled RTALLL as reference). Notably, HPLC–MS measurements revealed two different peaks with the same molecular mass, which turned out to be a transcyclization reaction in the linker between maleimide and the coupled cysteine moiety. With regard to the EGFR specificity, subsequent biological investigations (3-day viability, 14-day clonogenic assays and platinum uptake) on four different cell lines with different verified EGFR expression levels were performed. Unexpectedly, the results showed neither an enhanced activity nor an EGFR expression-dependent uptake of our new compounds. Consequently, fluorophore-coupled peptides were synthesized to re-evaluate the targeting ability of LARLLT itself. However, also with these molecules, flow cytometry measurements showed no correlation of drug uptake with the EGFR expression levels. Taken together, we successfully synthesized the first platinum(IV) complexes coupled to an EGFR-targeting peptide; however, the biological investigations revealed that LARLLT is not an appropriate peptide for enhancing the specific uptake of small-molecule drugs into EGFR-overexpressing cancer cells.Electronic supplementary materialThe online version of this article (doi:10.1007/s00775-017-1450-7) contains supplementary material, which is available to authorized users.
“…Its activity increases in the yeast Candida albicans during hyphal growth (36) and in S. cerevisiae during mating, which correlates with an increase in chitin formation (54). The enzyme is inhibited by UDP-GlcNAc in C. albicans (36), Drosophila melanogaster (20), and bacteria (26). Gfa1p activity is regulated by a protein kinase(s); the protein kinase A-dependent phosphorylated form of Gfa1p appears to have a higher activity than the unphosphorylated protein (20,57).…”
In Saccharomyces cerevisiae most chitin is synthesized by Chs3p, which deposits chitin in the lateral cell wall and in the bud-neck region during cell division. We have recently found that addition of glucosamine (GlcN) to the growth medium leads to a three-to fourfold increase in cell wall chitin levels. We compared this result to the increases in cellular chitin levels associated with cell wall stress and with treatment of yeast with mating pheromone. Since all three phenomena lead to increases in precursors of chitin, we hypothesized that chitin synthesis is at least in part directly regulated by the size of this pool. This hypothesis was strengthened by our finding that addition of GlcN to the growth medium causes a rapid increase in chitin synthesis without any pronounced change in the expression of more than 6,000 genes monitored with Affymetrix gene expression chips. In other studies we found that the specific activity of Chs3p is higher in the total membrane fractions from cells grown in GlcN and from mutants with weakened cell walls. Sucrose gradient analysis shows that Chs3p is present in an inactive form in what may be Golgi compartments but as an active enzyme in other intracellular membrane-bound vesicles, as well as in the plasma membrane. We conclude that Chs3p-dependent chitin synthesis in S. cerevisiae is regulated both by the levels of intermediates of the UDP-GlcNAc biosynthetic pathway and by an increase in the activity of the enzyme in the plasma membrane.
“…This enzyme is encoded by the gene gimS, which maps to 84 min on the Escherichia coli chromosome (39). It is a dimer of identical, 68-kDa subunits showing classic properties of amidotransferases (4,5,12,18). It is subject to weak product inhibition at millimolar concentrations of GlcN-6-P but is not subject to allosteric regulation (36,40), unlike the equivalent eukaryotic enzymes, which are allosterically inhibited by UDP-GlcNAc (for examples, see references 10, 17, 19, and 41).…”
The intracellular concentration of the enzyme glucosamine-6-phosphate synthase, encoded by the gene gimS in Escherichia coli, is repressed about threefold by growth on the amino sugars glucosamine and N-acetylglucosamine. This regulation occurs at the level of gimS transcription. It is not due just to the presence of intracellular amino sugar phosphates, because mutations which derepress the genes of the nag regulon (coding for proteins involved in the uptake and metabolism ofN-acetylglucosamine) also repress the expression ofglmS in the absence of exogenous amino sugars.
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