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At least 19 recordsLinked to original sources

PubMed1994

The Neurospora crassa chs-2 gene encodes a non-essential chitin synthase.

Chitin is a structural component of morphologically distinct structures assembled during various phases of growth and development in filamentous fungi. In Neurospora crassa, at least three different DNA fragments related to chitin synthase have been identified. In this study we cloned, sequenced and characterized the chitin synthase 2 structural gene (designated chs-2). The amino acid sequence deduced from the cloned chs-2 genomic DNA fragments is very similar to that of chitin synthase genes isolated from other fungi. Inactivation of the N. crassa chs-2 gene by repeat-induced point (RIP) mutation produced progeny which under standard growth conditions were indistinguishable from the wild-type. However, a significant reduction in chitin synthase activity and increased sensitivity to the phosphatidylcholine biosynthesis inhibitor edifenphos are characteristic of the chs-2RIP strain.

Amino Acid Sequence↗

PubMed2000

Identification of a novel inhibitor specific to the fungal chitin synthase. Inhibition of chitin synthase 1 arrests the cell growth, but inhibition of chitin synthase 1 and 2 is lethal in the pathogenic fungus Candida albicans.

As in Saccharomyces cerevisiae, the pathogenic fungus Candida albicans harbors three chitin synthases called CaChs1p, CaChs2p, and CaChs3p, which are structurally and functionally analogous to the S. cerevisiae ScChs2p, ScChs1p, and ScChs3p, respectively. In S. cerevisiae, ScCHS1, ScCHS2, and ScCHS3 are all non-essential genes; only the simultaneous disruption of ScCHS2 and ScCHS3 is lethal. The fact that a null mutation of the CaCHS1 is impossible, however, implies that CaCHS1 is required for the viability of C. albicans. To gain more insight into the physiological importance of CaCHS1, we identified and characterized a novel inhibitor that was highly specific to CaChs1p. RO-09-3143 inhibited CaChs1p with a K(i) value of 0.55 nm in a manner that was non-competitive to the substrate UDP-N-acetylglucosamine. RO-09-3143 also hampered the growth of the C. albicans cells with an MIC(50) value of 0.27 microm. In the presence of RO-09-3143, the C. albicans cells failed to form septa and displayed an aberrant morphology, confirming the involvement of the C. albicans Chs1p in septum formation. Although the effect of RO-09-3143 on the wild-type C. albicans was fungistatic, it caused cell death in the cachs2Delta null mutants but not in the cachs3Delta null mutants. Thus, it appears that in C. albicans, inhibition of CaChs1p causes cell growth arrest, but simultaneous inhibition of CaChs1p and CaChs2p is lethal.

Aminoglycosides↗

PubMed2003

Probing the mechanism of a fungal glycosyltransferase essential for cell wall biosynthesis. UDP-chitobiose is not a substrate for chitin synthase.

Chitin synthase is responsible for the biosynthesis of chitin, an essential component of the fungal cell wall. There is a long-standing question as to whether "processive" transferases such as chitin synthase operate in the same manner as non-processive transferases. The question arises from analysis of the polysaccharide structure--in chitin, for instance, each sugar residue is rotated approximately 180 degrees relative to the preceding sugar in the chain. This requires that the enzyme account for the alternating "up/down" configuration during biosynthesis. An enzyme with a single active site, analogous to the non-processive transferases--would have to accommodate a distorted glycosidic linkage at every other synthetic step. An alternative proposal is that the enzyme might assemble the disaccharide donor, addressing the "up/down" conformational problem prior to polymer synthesis. We present compelling evidence that this latter hypothesis is incorrect.

Cell Wall↗

PubMed2004

The first direct evaluation of the two-active site mechanism for chitin synthase.

Chitin synthase polymerizes UDP-GlcNAc to form chitin (poly-beta(1,4)-GlcNAc) and is essential for fungal cell wall biosynthesis. The alternating orientation of the GlcNAc residues within the chitin chain has led to the proposal that chitin synthase possesses two active sites. We report the results of the first direct test of this possibility. Two simple uridine-derived dimeric inhibitors are shown to exhibit 10-fold greater inhibition than a monomeric control, consistent with the presence of two active sites. This observation has important implications for the development of antifungal agents, as well as the understanding of polymerizing glycosyltransferases.

Binding Sites↗

PubMed1990

Chitin synthase I and chitin synthase II are not required for chitin synthesis in vivo in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the polysaccharide chitin forms the primary division septum between mother cell and bud. Two related enzymes, chitin synthase I and chitin synthase II (UDP-acetamido-2-deoxy-D-glucose:chitin 4-beta-acetamidodeoxyglucosyltransferase, EC 2.4.1.16), have been identified and their structural genes, CHS1 and CHS2, respectively, have been cloned and sequenced. Gene disruption experiments led to the conclusion that CHS2 is essential for cell division [Silverman, S.J., Sburlati, A., Slater, M.L. & Cabib, E. (1988) Proc. Natl. Acad. Sci. USA 85, 4735-4739], whereas CHS1 is not. We repeated the disruption of CHS2 and determined that it is not essential for vegetative growth. The viability of chs1::HIS3 chs2::TRP1 spores is influenced by strain background and germination conditions. The double disruption mutant has no detectable chitin deficiency in vivo, as judged by quantitative assay and by staining cells with Calcofluor. Assay of membrane preparations from the double disruption mutant indicates the presence of chitin synthetic activity. Unlike the CHS gene products, this third activity is not stimulated by trypsin. Characterization of the double disruption mutant revealed abnormalities in morphology and nuclear migration.

Blotting, Southern↗

PubMed2004

Second-generation dimeric inhibitors of chitin synthase.

Chitin synthase (CS) is essential for fungal cell wall biosynthesis and is an attractive medicinal target. Expanded results from our efforts to develop mechanism based inhibitors of CS are presented here. Specifically, we describe uridine dimers linked by tartrate amides as potential pyrophosphate mimics.

Antifungal Agents↗

PubMed1999

Chitin synthase genes in the arbuscular mycorrhizal fungus Glomus versiforme: full sequence of a gene encoding a class IV chitin synthase.

Chitin synthase genes of the arbuscular mycorrhizal fungus Glomus versiforme were sought in an investigation of the molecular basis of fungal growth. Three DNA fragments (Gvchs1, Gvchs2 and Gvchs3) corresponding to the conserved regions of distinct chitin synthase (chs) genes were amplified by means of the polymerase chain reaction (PCR) with two sets of degenerate primers. Gvchs1 and Gvchs2 encode two class I chitin synthases, whereas Gvchs3 encodes a class IV chitin synthase. A genomic library was used to obtain the Gvchs3 complete gene (1194 amino acids), which shows a very close similarity to the class IV chitin synthase from Neurospora crassa.

Amino Acid Sequence↗

PubMed2005

Iminosugars as glycosyltransferase inhibitors: synthesis of polyhydroxypyrrolidines and their evaluation on chitin synthase activity.

Chitin synthase is an enzyme involved in the biosynthesis of chitin, a major structural component of the cell wall of many fungi. Since chitin is absent in vertebrates, chitin synthase has been envisaged as a valuable target in the search for new antifungal agents. In this report, a series of C-2 substituted polyhydroxypyrrolidines were designed and synthesized with the aim of mimicking the glycosylation involved at the transition state of the enzymatic reaction governed by chitin synthase. Some of these models displayed chitin synthase inhibition in the millimolar range. However, no significant antifungal activity was noted on a panel of fungal strains.

Antifungal Agents↗

PubMed2003

CHS8-a fourth chitin synthase gene of Candida albicans contributes to in vitro chitin synthase activity, but is dispensable for growth.

In silico analysis of the genome sequence of the human pathogenic fungus Candida albicans identified an open reading frame encoding a putative fourth member of the chitin synthase gene family. This gene, named CaCHS8, encodes an 1105 amino acid open reading frame with the conserved motifs characteristic of class I zymogenic chitin synthases with closest sequence similarity to the non-essential C. albicans class I CHS2 gene. Although the CaCHS8 gene was expressed in both yeast and hyphal cells, homozygous chs8 Delta null mutants had normal growth rates, cellular morphologies and chitin contents. The null mutant strains had a 25% reduction in chitin synthase activity and were hypersensitive to Calcofluor White. A chs2 Delta chs8 Delta double mutant had less than 3% of normal chitin synthase activity and had increased wall glucan and decreased mannan but was unaffected in growth or cell morphology. The C. albicans class I double mutant did not exhibit a bud-lysis phenotype as found in the class I chs1 Delta mutant of Saccharomyces cerevisiae. Therefore, C. albicans has four chitin synthases with two non-essential class I Chs isoenzymes that contribute collectively to more than 97% of the in vitro chitin synthase activity.

Amino Acid Sequence↗

PubMed1986

The S. cerevisiae structural gene for chitin synthase is not required for chitin synthesis in vivo.

The chitin synthase of Saccharomyces is a plasma membrane-bound zymogen. Following proteolytic activation, the enzyme synthesizes insoluble chitin that has chain length and other physical properties similar to chitin found in bud scars. We isolated mutants lacking chitin synthase activity (chs1) and used these to clone CHS1. The gene has an open reading frame of 3400 bases and encodes a protein of 130 kd. The fission yeast S. pombe lacks chitin synthase and chitin. When a plasmid encoding a CHS1-lacZ fusion protein is introduced into S. pombe, both enzymatic activities are expressed in the same ratio as in S. cerevisiae, demonstrating that CHS1 encodes the structural gene of chitin synthase. Three CHS1 gene disruption experiments were performed. In all cases, strains with the disrupted gene have a recognizable phenotype, lack measurable chitin synthase activity in vitro but are viable, contain normal levels of chitin in vivo, and mate and sporulate efficiently.

Amino Acid Sequence↗

PubMed2005

Insect chitin synthases: a review.

Chitin is the most widespread amino polysaccharide in nature. The annual global amount of chitin is believed to be only one order of magnitude less than that of cellulose. It is a linear polymer composed of N-acetylglucosamines that are joined in a reaction catalyzed by the membrane-integral enzyme chitin synthase, a member of the family 2 of glycosyltransferases. The polymerization requires UDP-N-acetylglucosamines as a substrate and divalent cations as co-factors. Chitin formation can be divided into three distinct steps. In the first step, the enzymes' catalytic domain facing the cytoplasmic site forms the polymer. The second step involves the translocation of the nascent polymer across the membrane and its release into the extracellular space. The third step completes the process as single polymers spontaneously assemble to form crystalline microfibrils. In subsequent reactions the microfibrils combine with other sugars, proteins, glycoproteins and proteoglycans to form fungal septa and cell walls as well as arthropod cuticles and peritrophic matrices, notably in crustaceans and insects. In spite of the good effort by a hardy few, our present knowledge of the structure, topology and catalytic mechanism of chitin synthases is rather limited. Gaps remain in understanding chitin synthase biosynthesis, enzyme trafficking, regulation of enzyme activity, translocation of chitin chains across cell membranes, fibrillogenesis and the interaction of microfibrils with other components of the extracellular matrix. However, cumulating genomic data on chitin synthase genes and new experimental approaches allow increasingly clearer views of chitin synthase function and its regulation, and consequently chitin biosynthesis. In the present review, I will summarize recent advances in elucidating the structure, regulation and function of insect chitin synthases as they relate to what is known about fungal chitin synthases and other glycosyltransferases.

Amino Acid Sequence↗

PubMed1998

Molecular analysis of Chs3p participation in chitin synthase III activity.

Chitin is a minor but essential component of the cell wall of Saccharomyces cerevisiae, with functions in septum formation in the vegetative life cycle and also in conjugation and spore cell-wall synthesis in the sexual cycle. Of the three chitin synthases present in yeast, chitin synthase III (CSIII) is responsible for the synthesis of most of the chitin found in the cell, including a chitin ring at early budding, chitin interspersed in the cell wall, and chitin laid down during the sexual cycle. We have tagged Chs3p, the putative catalytic subunit of CSIII, with the immunoreactive epitope of influenza virus hemagglutinin to follow expression of the protein. Little correlation was found between the levels of transcription and translation of Chs3p and in vivo function, supporting our previous conclusion that regulation of CSIII occurs at the posttranslational level. To identify possible regions of the protein involved in catalysis or regulation, mutations were generated in the QRRRW 'signature sequence' of chitin synthases. Arginine residue mutations in Chs3p, and in Chs1p and Chs2p, resulted in a loss of both function in vivo and enzymatic activity. Mutations in a serine residue adjacent to glutamine in Chs3p caused loss of function in vivo with a moderate decrease in CSIII activity, suggesting a regulatory role for the serine residue in chitin biosynthesis. Several truncations in the unique hydrophilic carboxy-terminal region of Chs3p identified a sequence of about 25 amino acids that is required for both function and in vitro activity. Since this region is not present in Chs1 or Chs2, it may be involved in the specific regulation of CSIII.

Benzenesulfonates↗

PubMed1993

Identification of a Saccharomyces cerevisiae mutation that allows cells to grow without chitin synthase 1 or 2.

Chitin is a component of the yeast cell wall which is localized to the septum between mother and daughter cells. Previous work in Saccharomyces cerevisiae has shown that this organism possesses three chitin synthases, 1, 2, and 3. Disruption experiments have shown that loss of chitin synthase 2 has a more profound effect on cell viability than loss of either of the other two and is lethal in complete media. We report here the finding of an S. cerevisiae strain which does not require the chitin synthase 2 structural gene for viability. We present evidence that there is a gene in this strain which suppresses the lethality of disruption of the chitin synthase 2 structural gene and is genetically distinct from the structural genes for chitin synthase 1 and chitin synthase 2. We show that an S. cerevisiae mutant containing the suppressor and lacking both structural genes for chitin synthase 1 and 2 has normal amounts of chitin in its cell wall. We hypothesize that the suppressor gene encodes or controls the expression of chitin synthase 3.

Antifungal Agents↗

PubMed2006

Prenylation of Saccharomyces cerevisiae Chs4p Affects Chitin Synthase III activity and chitin chain length.

Chs4p (Cal2/Csd4/Skt5) was identified as a protein factor physically interacting with Chs3p, the catalytic subunit of chitin synthase III (CSIII), and is indispensable for its enzymatic activity in vivo. Chs4p contains a putative farnesyl attachment site at the C-terminal end (CVIM motif) conserved in Chs4p of Saccharomyces cerevisiae and other fungi. Several previous reports questioned the role of Chs4p prenylation in chitin biosynthesis. In this study we reinvestigated the function of Chs4p prenylation. We provide evidence that Chs4p is farnesylated by showing that purified Chs4p is recognized by anti-farnesyl antibody and is a substrate for farnesyl transferase (FTase) in vitro and that inactivation of FTase increases the amount of unmodified Chs4p in yeast cells. We demonstrate that abolition of Chs4p prenylation causes a approximately 60% decrease in CSIII activity, which is correlated with a approximately 30% decrease in chitin content and with increased resistance to the chitin binding compound calcofluor white. Furthermore, we show that lack of Chs4p prenylation decreases the average chain length of the chitin polymer. Prenylation of Chs4p, however, is not a factor that mediates plasma membrane association of the protein. Our results provide evidence that the prenyl moiety attached to Chs4p is a factor modulating the activity of CSIII both in vivo and in vitro.

Chitin↗

PubMed1987

Chitin synthase activity and the rate of chitin formation in cell-division cycle mutant Saccharomyces cerevisiae cdc 24.

At the nonpermissive temperature (37 degrees C) the cells of the temperature-sensitive mutant Saccharomyces cerevisiae cdc 24 accumulated chitin 10 times faster than at 22 degrees C. In situ determinations of the activity of chitin synthase revealed that in the cells grown at 37 degrees C more than 37% of the total chitin synthase were in the active state whereas in cells grown at 22 degrees C only 7% of the potential enzyme activity were expressed. When the enzyme activity was calculated per cell number unit, there was 10 times more of the active chitin synthase per cell in the cells grown at 37 degrees C than in the cells grown at 22 degrees C, a value which correlated well with the observed difference in the rates of chitin accumulation at different temperatures.

Chitin↗

PubMed1994

Characterization of chitin synthase from Botrytis cinerea.

Chitin synthase in a microsomal preparation from Botrytis cinerea had an apparent Km for UDP-N-acetylglucosamine of 2.0 mM while nikkomycin Z and polyoxin D inhibited enzyme activity competitively with apparent Ki values of approximately 0.1 microM and 6 microM respectively. The organophosphorus fungicide edifenphos was a non-competitive inhibitor (Ki(app) 54 microM). Preincubation of microsomes for 2 h at 25 degrees C resulted in a maximum twofold stimulation of chitin synthase activity while preincubation with trypsin (25 micrograms ml-1) or cytosol (350 micrograms cytosolic protein ml-1) for 10 min at 25 degrees C resulted in approximately fourfold and 20-fold increases in chitin synthase activity, respectively. A range of protease inhibitors reduced the degree of activation of microsomal chitin synthase by cytosol. Most potent were phenylmethanesulphonyl fluoride and chymostatin; these compounds completely inhibited activation of enzyme activity. Two fragments (approx. 600 bp; CHS1 and CHS2) were amplified from B. cinerea genomic DNA using degenerate PCR primers based on regions of complete amino acid homology between previously published chitin synthase gene sequences. When the DNA and predicted amino acid sequences of CHS1 were used to probe computer databases for related sequences, B. cinerea CHS1 was found to be most similar to CHS1 from Neurospora crassa.

Amino Acid Sequence↗

PubMed2003

Chitin synthase III activity, but not the chitin ring, is required for remedial septa formation in budding yeast.

Chitin is a minor but essential component of the Saccharomyces cerevisiae cell wall. In wild-type, chitin synthase II is required for the formation of primary septa and chitin synthase III (CSIII) is not essential. However, in chs2 mutants CSIII becomes essential for the formation of aberrant septa. We examined which of two CSIII functions, the formation of a chitin ring at bud emergence or of chitin in the remedial septa, was required for viability. By using cell cycle synchronization in combination with nikkomycin Z, a specific inhibitor of CSIII, we inhibited chitin synthesis in a chs2 mutant, during formation of either the ring or the remedial septa. The results show that only synthesis of the chitin during aberrant septa formation is essential for viability. Thus, the unique function of the chitin ring seems to be maintenance of the integrity of the mother-bud neck, as we recently found, and the importance of chitin in septum closure, both in normal and abnormal situations, is underlined.

Aminoglycosides↗

PubMed1989

Chitin synthase 1, an auxiliary enzyme for chitin synthesis in Saccharomyces cerevisiae.

Previously, we showed that chitin synthase 2 (Chs2) is required for septum formation in Saccharomyces cerevisiae, whereas chitin synthase 1 (Chs1) does not appear to be an essential enzyme. However, in strains carrying a disrupted CHS1 gene, frequent lysis of buds is observed. Lysis occurs after nuclear separation and appears to result from damage to the cell wall, as indicated by osmotic stabilization and by a approximately 50-nm orifice at the center of the birth scar. Lysis occurs at a low pH and is prevented by buffering the medium above pH 5. A likely candidate for the lytic system is a previously described chitinase that is probably involved in cell separation. The chitinase has a very acidic pH optimum and a location in the periplasmic space that exposes it to external pH. Accordingly, allosamidin, a specific chitinase inhibitor, substantially reduced the number of lysed cells. Because the presence of Chs1 in the cell abolishes lysis, it is concluded that damage to the cell wall is caused by excessive chitinase activity at acidic pH, which can normally be repaired through chitin synthesis by Chs1. The latter emerges as an auxiliary or emergency enzyme. Other experiments suggest that both Chs1 and Chs2 collaborate in the repair synthesis of chitin, whereas Chs1 cannot substitute for Chs2 in septum formation.

Acetylglucosamine↗