Glucan Synthetase Research Articles

Ibrexafungerp: First Approval.

Ibrexafungerp (BREXAFEMME®) is an orally active triterpenoid antifungal drug being developed by SCYNEXIS, Inc. for the treatment of fungal infections. The inhibition of β-1,3-D glucan synthetase by ibrexafungerp compromises the integrity of fungal cell walls. Ibrexafungerp has been recently approved for the treatment of vulvovaginal candidiasis (VVC), and it is the first novel antifungal drug class to be approved in more than 20 years. Development for the treatment of recurrent VVC and invasive fungal infections is ongoing. This article summarizes the milestones in the development of ibrexafungerp leading to this first approval for the treatment of VVC in adult and postmenarchal paediatric females.

ChemInform Abstract: Carbohydrates as Structural Constituents of Yeast Cell Wall and Septum

The cell walls and septa of fungi a re useful models for morphogenesis and potential targets for anti fungal agents. In the yeast, Saccharomvces cerevisiae, the main structural components of the primary septum and of the cell wall are chitin and ,¶(1+3)glucan, respectively. Two chitin synthetases have been identified in yeast, one of which is essential for septum formation, whereas the other one has a repair function. in extracts in a zymogenic form that can be activated by proteases. /?(1-+3)Glucan synthetase is also attached to the plasma membrane. activity is strongly stimulated by GTP and its analogs. extraction of the membranes, two components have been obtained

Pathogenic Fungi—Structural Biology and Taxonomy

Structural Biology and Taxonomy is the first volume in a series authored by leading medical mycologists. The series' scope is to review progress in basic research on zoopathogenic fungi, a timely effort as medical mycology moves into the genomics era. General knowledge of fungal pathogens is assumed. This volume begins by discussing the cell wall which, besides its roles in pathogenesis, is now a practical drug target. The molecular architecture of fungal walls remains elusive because of difficulty in correlating chemical composition with the ultrastructural layers and uncertainty concerning the linkages connecting major cell wall polymers: glucans, mannan, and chitin. The basis for morphogenesis is the holy grail of medical mycology because temperature-sensitive dimorphism is a stratagem used by several deep-seated fungal pathogens. The fungal cell cyle is considered with respect to the mechanism of sequential gene expression in Candida albicans, since little is known about the cell cycle in pathogenic molds. Important interpretation is provided about the hyphal form of C. albicans, which clarifies the germ tube's role in morphogenesis and, potentially, in disease. The molecular genetics of morphogenesis in C. albicans follows. Hyphal during infection is arguably a pathogenic factor since it thwarts phagocytosis. Genes controlling hyphal development include ones that are upregulated during cell elongation and adherence to epithelia. Fine tuning of morphogenesis is illustrated by the enhanced filamentous growth gene which, when knocked out, blocks the transition to the mycelial form. Early steps in this transition are complex, with at least 2 signaling pathways identified: 1 stops yeast and another, with a heat shock protein 70-type profile, initiates the assembly of proteins necessary for mycelial growth. As the focus on morphogenesis continues, dimorphism in several endemic mycoses is concisely reviewed. Heat shock proteins are emphasized because of the temperature-sensitive morphogenesis to the tissue form. Yeast-form–specific genes identified in Histoplasma capsulatum function in calcium/calmodulin signaling pathways and sulphur metabolism. Calcium-dependent signaling pathways and heat shock protein expression regulate dimorphism in Paracoccidioides brasiliensis and have broad implications for other pathogens. Coccidioides immitis dimorphism involves the construction and rupture of the spherule, or tissue form, for which chitin and glucan synthetases and hydrolases are key enzymes. A further chapter discusses how surface membrane G-protein receptors in C. albicans and other fungi transmit external stimuli through 2 major protein kinase cascades. These govern multiple functions, including hyphal development and the secretion of pathogenic factors. Departing from the morphogenesis theme, chapters on phylogenetic analysis help clarify the taxonomy of noncultivatable medical fungi (excluding Pneumocystis). The evolution and population genetics of 3 agents of major deep-seated mycoses, which follow, are of more general interest. Finally, in a return to the original cell wall theme, the fine structure of mannans and galactomannans is explained as a useful adjunct in classifying fungi. Pathogenic Fungi—Structural Biology and Taxonomy is an important interpretation of recent literature, a valuable addition to collections, and recommended reading for investigators seeking a broad appreciation for the current state of the art.

Molecular Cloning and Characterization of cgs , the Brucella abortus Cyclic β(1-2) Glucan Synthetase Gene: Genetic Complementation of Rhizobium meliloti ndvB and Agrobacterium tumefaciens chvB Mutants

The animal pathogen Brucella abortus contains a gene, cgs, that complemented a Rhizobium meliloti nodule development (ndvB) mutant and an Agrobacterium tumefaciens chromosomal virulence (chvB) mutant. The complemented strains recovered the synthesis of cyclic beta(1-2) glucan, motility, virulence in A. tumefaciens, and nitrogen fixation in R. meliloti; all traits were strictly associated with the presence of an active cyclic beta(1-2) glucan synthetase protein in the membranes. Nucleotide sequencing revealed the presence in B. abortus of an 8.49-kb open reading frame coding for a predicted membrane protein of 2,831 amino acids (316.2 kDa) and with 51% identity to R. meliloti NdvB. Four regions of the B. abortus protein spanning amino acids 520 to 800, 1025 to 1124, 1284 to 1526, and 2400 to 2660 displayed similarities of higher than 80% with R. meliloti NdvB. Tn3-HoHo1 mutagenesis showed that the C-terminal 825 amino acids of the Brucella protein, although highly conserved in Rhizobium, are not necessary for cyclic beta(1-2) glucan synthesis. Confirmation of the identity of this protein as B. abortus cyclic beta(1-2) glucan synthetase was done by the construction of a B. abortus Tn3-HoHo1 insertion mutant that does not form cyclic beta(1-2) glucan and lacks the 316.2-kDa membrane protein. The recovery of this mutant from the spleens of inoculated mice was decreased by 3 orders of magnitude compared with that of the parental strain; this result suggests that cyclic beta(1-2) glucan may be a virulence factor in Brucella infection.

Comparative characteristics of proteins of cottonplant lines differing in the strength of the fiber

The enzymes and proteins of the fibers of two lines of cotton plant differing in the strength of the fiber have been investigated. It has been shown that the activities of glucan synthetase and peroxidase rise as the fiber matures, while the activities of β-(1-3)-glucanase and cellulase fall. The specific enzymatic activities of peroxidase and glucan synthetase in the L-175 line, distinguished by a stronger fiber, are higher than for the L-466 line with a weaker fiber. The activity of glucanase changes according to the strength of the fiber. In a study of the protein composition of cotton fibers, polypeptides with molecular masses of 28 and 39 kDa were found among the proteins responsible for the strength of the fiber.

Action de la désertomycine sur la synthèse des polymères pariétaux du Saccharomyces uvarum

The desertomycin action upon Saccharomyces uvarum wall synthesis has been studied. Spheroblast regeneration was carried out in a liquid medium containing labeled glucose to monitor the synthesis of different wall components. In the presence of desertomycin, wall synthesis was affected; this was expressed as a net reduction of insoluble alkali constituents content, more precisely the insoluble acido-alkali fraction that, in yeasts, is constituted by chains of beta(1,3)-glucans linked among themselves by beta(1,6) bonds. Mannan formation was not inhibited such polymers that cannot be fixed to the glucan matrix of the wall were liberated in the regeneration medium. Because of desertomycin action, the decrease in insoluble alkali content revealed an interference with the enzymatic systems catalyzing glucan synthesis. In vitro, however, this antifungal had little effect upon glucan synthetase activity: doses 5 times superior to the subinhibiting level used in vivo caused only 30% inhibition. This result can be explained by an indirect action of desertomycin. Parietal disorders were the result of membrane structure disturbance, notably the phospholipids and localized enzymatic systems. This antifungal presents an analogical structure with macrolides with recognized membrane action.

Kinetic characterization of the synthesis of cotton cellulose

The influence of the temperature, pH, and inhibitors on the synthesis of cellulose from cottonplant shoots has been investigated. The maximum activity of glucan synthetase was exhibited at 27°C, pH 8. O. The greatest inhibition of the formation of cellulose was shown by EDTA.

Lipid composition and proton transport in Penicillium cyclopium and Ustilago maydis plasma membrane vesicles isolated by two-phase partitioning

Plasma membranes have been isolated and purified from two species of fungi, Penicillium cyclopium and Ustilago maydis, using a two-phase aqueous polymer technique. The membranes were characterised using marker enzyme assays (e.g., vanadate-sensitive (Mg 2+-K +)-ATPase and glucan synthetase II) and lipid composition (sterol enrichment, increased phosphatidylethanolamine/phosphatidylcholine ratio, and the absence of diphosphatidylglycerol). The proton-pumping activities of the plasma membrane-bound H +-ATPases from these species were compared. H +-ATPase activity was found to be greater in U. maydis than in P. cyclopium, which was attributed to differences in orientation of the plasma membrane vesicles. There was evidence to suggest the presence of redox chain activity in the plasma membranes of both species.

Isolation of plasma membranes from mung bean hypocotyl sections by two‐phase partitioning: suitability of the technique for ageing tissues

This paper discusses the application of a particular two‐phase partitioning system to the isolation of plasma membranes from heterogeneous starting material, differing in physiological age. Plasma membranes were isolated from hypocotyl segments of mung beans (Vigna radiata L. Wilczek) on four successive days in order to examine the variation caused by ageing of the seedling. Additionally, the segments were cut at different positions of the hypocotyl to measure variation caused by position‐related ageing. To assess purity and degree of contamination of the plasma membrane‐enriched preparations, a series of membrane enzyme markers were screened for all isolated fractions. Glucan synthetase II activities were enriched in the plasma membrane fractions, but enrichment and recovery became less pronounced with increasing age. Plasma membrane ATPase activity affected by VO43‐, Ca2+ and K+ was similar in all segments throughout the time‐course of the experiment (4 days). However, control ATPase activity varied with segment origin: the physiologically oldest segments showed only 75% activity compared to the youngest ones. Km and Vmax values indicated a smaller proportion of active enzyme but higher substrate affinity as the age of the segments increased. Contamination by intracellular membranes was minimal and unrelated to tissue age.

Isolation of ?-(1?4)-glucan synthetase from cottonplant shoots

An enzyme complex including β-(1→4)-glucan synthetase has been isolated from cottonplant shoots by chromatography and electrophoresis. Accrding to electrophoretic analysis under denaturing conditions, the molecular masses of the polypeptides presumably present in in the glucan synthetase complex amounted to 74, 50, and 30 kDa.

Localization of β-glucan synthetase in membranes ofParacoccidioides brasiliensis

To localize β-1,3-glucan synthetase in membranes of Paracoccidioides brasiliensis, particulate preparations were obtained from both phases of the fungus and separated on sucrose gradients. The yeast glucan synthetase distributed between the 20% and 30% sucrose bands whereas the mycelial enzyme concentrated in the 40% band. In all cases, vanadate-sensitive, azide-insensitive ATPase was found together with glucan synthetase, suggesting that the latter is located in the cytoplasmic membrane.

Alterations in the cell wall ofSaccharomyces cerevisiae induced by the alpha sex factor or a mutation in the cell cycle

We performed experiments in parallel to study the rate of synthesis of cell wall polysaccharides and the activity of glycosyl transferases in Saccharomyces cerevisiae after arrest of a cdc 28 mutant in G1 phase by either addition of alpha-factor or transfer to the non-permissive temperature. Both effectors brought about similar time-dependent increases in the rate of synthesis and deposition of the cell wall polysaccharides chitin, glucan and mannan. These changes in cell wall composition were accompanied by an increase in the specific activities of glucan and chitin synthetases. This increase was inhibited by cycloheximide suggesting that it represented de novo enzyme biosynthesis and not enzyme activation. Our data are consistent with the notion that both alpha-factor and the cdc 28 mutation affect the same stage-specific function that controls the temporal expression of glycosyl transferases.

Biosynthesis of ?-glucans in fungi

Glucans are the most abundant polysaccharides present in fungi. The present review provides updated information on the structure and synthesis of beta-glucans in fungal cells. Synthesis of these polymers made up of B1,3 chains with a variable degree of B1,6 branching involves several reactions: initiation, chain elongation and branching, of which the most studied one is the elongation step. This reaction, catalyzed by the so-called glucan synthetases, utilizes UDPG as sugar donor. Properties of glucan synthetases are extremely variable depending on the fungal species, and their developmental stage. Because of the importance of these polysaccharides it is anticipated that comprehension of their mechanism of synthesis, is important for the understanding of cell wall assembly and cell growth and morphogenesis, as well as for the design of specific antifungal drugs.

Peptide sequences for sucrose splitting and glucan binding within Streptococcus sobrinus glucosyltransferase (water-insoluble glucan synthetase)

The gene encoding glucosyltransferase responsible for water-insoluble glucan synthesis (GTF-I) of Streptococcus sobrinus (formerly Streptococcus mutans 6715) was cloned, expressed, and sequenced. A gene bank from S. sobrinus 6715 DNA was constructed in vector pUC18 and screened with anti-GTF-I antibody to detect clones producing GTF-I peptide. Five immunopositive clones were isolated, all of which produced peptides that bound alpha-1,6 glucan. GTF-I activity was found in only two large peptides: one stretching over the full length of the GTF-I peptide and composed of about 1,600 amino acid residues (AB1 clone) and the other lacking about 80 N-terminal residues and about 260 C-terminal residues (AB2 clone). A deletion study of the AB2 clone indicated that specific glucan binding, which is essential for water-insoluble glucan synthesis, was lost prior to sucrase activity with an increase in deletion from the 3' end of the GTF-I gene. These results suggest that the GTF-I peptide consists of three segments: that for sucrose splitting (approximately 1,100 residues), that for glucan binding (approximately 240 residues), and that of unknown function (approximately 260 residues), in order from the N terminus. The primary structure of the GTF-I peptide, deduced by DNA sequencing of the AB1 clone, was found to be very similar to that of the homologous protein from another strain of S. sobrinus.

Carbohydrates as structural constituents of yeast cell wall and septum

Abstract

Senescence of oat leaves: changes in the free sterol composition and enzyme activities of the plasma membrane

Plasma membrane fractions were prepared from young (10-day-old), mature (17-day-old) and senescent (10 + 7-day-old) oat leaves ( Avena sativa L.) through aqueous two-phase partitioning and analyzed for their free sterol composition. Continued leaf growth resulted in a strong increase in overall sterol content from 40 nmol · mg −1 lipid to about 120 nmol · mg −1 lipid (1.5% and 5% by wt. of total lipids respectively). This increase could be completely ascribed to increased cholesterol and stigmasterol concentrations. Kinetic parameters of vanadate-sensitive ATPase and NADH-FeCN reductase activities did not change significantly with leaf maturation. The sterol concentration increased to a lesser extent (4.2% and 4.4% by weight) in the plasma membrane of 10-day-old leaf segments that were cut and kept in light or dark for 7 more days. However, the increase in this case was completely due to a raise in Δ 7-avenasterol and 28-isofucosterol concentrations. The K m and V max values for the ATPase activity decreased significantly in these membranes. The characteristics of the FeCN 2 reductase and of glucan synthetase II activity were apparently not affected. The effect of senescence on sterol biosynthesis and on plasma membrane enzyme activities is discussed.

Paracoccidioides brasiliensis: Current Research on Morphogenesis

The involvement of cell wall glucans in the process of dimorphism in Paracoccidioides brasiliensis has been proposed for many years. The control of glucan synthetase activity through cytoplasmic factors is also considered. Current research on biochemical aspects of dimorphism in P. brasiliensis suggests that proteinases are involved in the control of β-glucan synthetase activity in the generation of the fungal cell wall. The probable involvement of protein disulfide reductase is also suggested. A complementary model of yeast mycelium transformation is proposed.

Isolation and characterization of right-side-out plasma membrane vesicles from coleoptiles of Zea mays L. using reorientating sucrose gradients

Reorientating continuous sucrose gradients in a vertical rotor were used for rapid isolation of right-side-out sealed membrane vesicles from corn coleoptiles with high purity. Membranes were identified as plasma membrane vesicles by vanadate-sensitive ATPase, glucan synthetase II, NADH-K 3Fe(CN) 6-oxidoreductase activities and a dithionite-reducible b-type cytochrome. A molybdate-resistant acid phosphatase was associated with the isolated plasma membrane fraction; this was characterized and differentiated from the soluble vacuolar enzyme. Plasma membrane vesicles contained high amounts of the cytosolic marker enzyme alcohol dehydrogenase. This enzyme was only detectable in the presence of 0.03% Triton X-100, indicating that vesicles originated from the initial homogenization and remained right-side-out during all preparation procedures.

Direct isolation of vacuoles from leaf tissue of lettuce (Lactuca sativa) retaining protoplasts within the leaves

A procedure is described in which vacuoles are isolated from leaf tissue of lettuce (Lactuca sativa L.). After incubation in an enzyme solution, the vacuoles are directly extracted from the leaf tissue by osmotic shock using a phosphate buffer. In this method no protoplasts are released from the leaf tissue. This procedure avoids the problems of separating vacuoles from protoplasts with similar density. To evaluate the purity of the vacuoles, the activity of glucan synthetase 11 (EC 2.4.1.34), NAD(P) H-cytochrome c reductase (EC 1.6.99.3) and malate dehydrogenase (EC 1.1.1.37) was measured. To measure vanadate- and nitrate-sensitive ATPase activity (EC 3.6.1.8) vesicles were prepared from the vacuoles and ATP-dependent vesicle acidification was measured as acridine orange quenching. Nitrate inhibited the quenching, while addition of vanadate had no effect. It was concluded that the vacuoles were not contaminated with plasma membranes. To evaluate the viability of the vacuoles [(14) C]-malate uptake was measured. The vacuoles showed a constant rate of [(14) C]-malate uptake during 45 min. This rate was maximal at pH 6.8.

Plasma membrane biogenesis in higher plants: In vivo transfer of lipids to the plasma membrane

The intermembrane transfer of lipids and particularly of very long chain fatty acids (VLCFA) from a light membrane fraction (1.08–1.09 g/cm 3) to By means of biochemical markers (glucan synthetase II, IDPase, CDP-choline diglyceride transferase and NADPH cyt c reductase), it was shown that th The biosynthesis of VLCFA was assayed in vitro in the purified plasma membrane and the other membranes after phase partition of the microsomal pell