Browsing by Author "Nguyen, Thi Thuy Minh"
Now showing 1 - 2 of 2
Results Per Page
Sort Options
Item Influence of inhibitors of sterol biosynthesis on growth and sterol metabolism in Candida albicans(Texas Tech University, 2003-08) Nguyen, Thi Thuy MinhCandida albicans was treated with seven antifungal agents designed to inhibit ergosterol biosynthesis. Two types of inhibitors were studied based on their hypothetical mode of action: mechanism-based inactivators, containing either a sulfur or bromine in the sterol side chain and transition state mimics, which contain a nitrogen in the sterol side chain or in the nucleus. The sterol composition of cells treated with the various compounds was determined using three chromatographic techniques, thin-layer chromatography, reverse-phase high-performance liquid chromatography and gas chromatography-mass spectrometry. Additional characterization of the sterol identities was by proton nuclear magnetic resonance spectroscopy and ultra-violet spectroscopy. Ergosterol was found to be the major sterol in control cultures (69%). In treated cells, the dominant effect on sterol biosynthesis was a significant decrease in the amount of ergosterol. The influence of containing a heteroatom in the side chain on sterol methyl transferase activity was evident in aberrant cell morphology of treated cells and a reduction in ergosterol production. Disturbance of the 24-alkyl to 24-desaikyi ratio by as little as 10 % is sufficient to elicit a fimgistatic response. A second class of inhibitor represented by 3p-aminoianosteroi, designed to inhibit the C-4 demethyiation pathway, was found to inhibit growth by impairing lanosterol metabolism. The relationship between ergosterol biosynthesis inhibition action on fungal growth and ergosterol homeostasis is discussed.Item Studies on Protein Engineered Soybean Sterol Methyltransferase to Probe the Structure-Function relationship of enzyme activities(Texas Tech University, 2009-08) Nguyen, Thi Thuy Minh; Nes, William David; Shaw, Robert W.; Pare, PaulSterol C24-methyltransferase (SMT) enzymes convert Δ24-sterol acceptor molecules to one or more methyl products and set the pattern of 24-alkyl sterols used as membrane inserts in plants. For this study, a series of cycloartenol-based acceptors that differed in the nature of the side chain construction were evaluated as substrates of the cloned soybean (Glycine max) SMT. Three natural substrates were tested with the soybean SMT1, cycloartenol (CA), cyclobranol (24-methyl cycloartenol, CB) and 24(28)-methylene cycloartanol (MCA); CA converted to a single product MCA, CB converted to a novel Δ24(28)-C25-methyl sterol and MCA converted to a triplet of 24-ethyl(idene) sterols. When these substrates were tested against a panel of soybean mutants designed using the results of the yeast SMT recently found to generate plant-like C1- or C2-transfer activity distinct differences were observed in product distributions between the plant and fungal SMTs. On the one hand, new transalkylation activities catalyzed by the fungal SMT assayed with zymosterol were not carried out by the plant SMT mutants assayed with CA. On the other hand, Tyr to Phe soybean SMT mutants at positions 81 and 223 (ERG6 nomenclature) generated novel products from assay with CB. A new substrate analog 26-difluorocycloartenol (diFCA) was studied to understand further the reaction mechanism catalyzed by the soybean SMT1. By use of fluorine as an isosteric replacement for a hydrogen atom in the sterol side chain, it was expected that the inductive electron-withdrawing effect of the fluoro substituent would impair the critical proton transfer of H24 to C25 affording covalent attachment of the enzyme-generated intermediate to the recombinant protein. Steady-state kinetic experiments with CA and diFCA showed that the overall catalytic efficiencies (Vmax/KM) differ markedly with the diFCA substrate much less efficient than the CA. The diFCA was found to be a competitive inhibitor of CA to MCA conversion catalyzed by the soybean SMT1 exhibiting a KI of 71 µM. In addition, conversion of the diFCA was evident in the GC-MS analysis of the enzyme-generated products which showed C24-methylated isomeric compounds of molecular weight 458 and 476 amu. These sterol derivatives were assigned the tentative structures of the monol derivatives (turnover products that possess 1 or 2 fluorine atoms at C26) and diol derivatives (kill products bound to the enzyme and released by saponification of the enzyme extract). This work affords compelling evidence in support of a cationic mechanism for C24-methylation of the cycloartenol side chain and that variant side chain features recognized by wild-type and mutant soybean SMT1 can affect catalytic competence, product diversity and/or enzyme inhibition.