Functional and structural properties of several truncated or mutated variants of Candida albicans Gfa1p (glucosamine-6-phosphate synthase) were compared with those of the wild-type enzyme. Fragments encompassing residues 1-345 and 346-712 of Gfa1p, expressed heterogeneously in bacterial host as His6 fusions, were identified as the functional GAH (glutamine amidehydrolysing) and ISOM (hexose phosphate-isomerizing) domains respectively. It was found that the native GAH domain is monomeric, whereas the native ISOM domain forms tetramers, as does the whole enzyme. Spectrofluorimetric and kinetic studies of the isolated domains, the Delta218-283Gfa1p mutein and the wild-type enzyme revealed that the binding site for the feedback inhibitor, uridine 5'-diphospho-N-acetyl-D-glucosamine, is located in the ISOM domain. Inhibitor binding affects amidohydrolysing activity of the GAH domain and, as a consequence, the GlcN-6-P (D-glucosamine-6-phosphate)-synthetic activity of the whole enzyme. The fragment containing residues 218-283 is neither involved in ligand binding nor in protein oligomerization. Comparison of the catalytic activities of Gfa1p(V711F), Delta709-712Gfa1p, Gfa1p(W97F) and Gfa1p(W97G) with those of the native Gfa1p and the isolated domains provided evidence for an intramolecular channel connecting the GAH and ISOM domains of Gfa1p. The channel becomes leaky upon deletion of amino acids 709-712 and in the W97F and W97G mutants. The Trp97 residue was found to function as a molecular gate, opening and closing the channel. The W97G and V711F mutations resulted in an almost complete elimination of the GlcN-6-P-synthetic activity, with the retention of the amidohydrolase and sugar phosphate-isomerizing activities. 相似文献
Development of effective and safe therapeutic treatment of fungal infections remains one of the major challenge for modern medicine. The aim of presented investigation was to analyze the in vitro antifungal activity of selected essential oils, ethanolic extracts of propolis and silver nanoparticles dropped on TiO2 against azole-resistant C. albicans (n = 20), C. glabrata (n = 14) and C. krusei (n = 10) clinical isolates. Among tested essential oils, the highest activity has definitely been found in the case of the oil isolated from the bark of Cinnamomum cassia, with MIC and MFC values for all tested strains in the range of 0.0006–0.0097 % (v/v) and 0.0012–0.019 % (v/v), respectively. High activity was also observed for the Lemon, Basil, Thyme, Geranium and Clove (from buds) essential oils. Significant differences in fungicidal activity have been observed in the case of four tested propolis samples. Only one of them revealed high activity, with MFC values in the range from 0.156 to 1.25 % (v/v). Satisfactory fungicidal activity, against C. albicans and C. glabrata isolates, was also observed in the case of silver nanoparticles, however C. krusei isolates were mostly resistant. We also revealed that constituents of most of essential oils and propolis as well as silver nanoparticles are not substrates for drug transporters, which belong to the most important factors affecting resistance of Candida spp. clinical isolates to many of conventional antimycotics. To conclude, the results of our investigation revealed that essential oils, propolis and silver nanoparticles represent high potential for controlling and prevention candidiasis. 相似文献
The plasticity of root architecture is crucial for plants to acclimate to unfavourable environments including low nitrogen (LN) stress. How maize roots coordinate the growth of axile roots and lateral roots (LRs), as well as longitudinal and radial cell behaviours in response to LN stress, remains unclear. Maize plants were cultivated hydroponically under control (4 mm nitrate) and LN (40 μm ) conditions. Temporal and spatial samples were taken to analyse changes in the morphology, anatomical structure and carbon/nitrogen (C/N) ratio in the axile root and LRs. LN stress increased axile root elongation, reduced the number of crown roots and decreased LR density and length. LN stress extended cell elongation zones and increased the mature cell length in the roots. LN stress reduced the cell diameter and total area of vessels and increased the amount of aerenchyma, but the number of cell layers in the crown root cortex was unchanged. The C/N ratio was higher in the axile roots than in the LRs. Maize roots acclimate to LN stress by optimizing the anatomical structure and N allocation. As a result, axile root elongation is favoured to efficiently find available N in the soil. 相似文献
Glucosamine-6-phosphate (GlcN-6-P) synthase catalyses the first and practically irreversible step in hexosamine metabolism.
The final product of this pathway, uridine 5’ diphospho N-acetyl-D-glucosamine (UDP-GlcNAc), is an essential substrate for
assembly of bacterial and fungal cell walls. Moreover, the enzyme is involved in phenomenon of hexosamine induced insulin
resistance in type II diabetes, which makes it a potential target for antifungal, antibacterial and antidiabetic therapy.
The crystal structure of the isomerase domain of GlcN-6-P synthase from human pathogenic fungus Candida albicans, in complex with UDP-GlcNAc has been solved recently but it has not revealed the molecular mechanism of inhibition taking
place under UDP-GlcNAc influence, the unique feature of the eukaryotic enzyme. UDP-GlcNAc is a physiological inhibitor of
GlcN-6-P synthase, binding about 1 nm away from the active site of the enzyme. In the present work, comparative molecular
dynamics simulations of the free and UDP-GlcNAc-bounded structures of GlcN-6-P synthase have been performed. The aim was to
complete static X-ray structural data and detect possible changes in the dynamics of the two structures. Results of the simulation
studies demonstrated higher mobility of the free structure when compared to the liganded one. Several amino acid residues
were identified, flexibility of which is strongly affected upon UDP-GlcNAc binding. Importantly, the most fixed residues are
those related to the inhibitor binding process and to the catalytic reaction. The obtained results constitute an important
step toward understanding of mechanism of GlcN-6-P synthase inhibition by UDP-GlcNAc molecule. 相似文献