Isolation of a gene involved in 1,3-beta-glucan synthesis in Aspergillus nidulans and purification of the corresponding protein

Abstract

Saccharomyces cerevisiae has two highly homologous genes, FKS1 and FKS2, which encode interchangeable putative catalytic subunits of 1,3-␤-glucan synthase (GS), an enzyme that synthesizes an essential polymer of the fungal cell wall. To determine if GS in Aspergillus species is similar, an FKS homolog, fksA, was cloned from Aspergillus nidulans by cross-hybridization, and the corresponding protein was purified. Sequence analysis revealed a 5,716-nucleotide coding region interrupted by two 56-bp introns. The fksA gene encodes a predicted peptide of 229 kDa, FksAp, that shows a remarkable degree of conservation in size, charge, amino acid identity, and predicted membrane topology with the S. cerevisiae FKS proteins (Fksps). FksAp exhibits 64 and 65% identity to Fks1p and Fks2p, respectively, and 79% similarity. Hydropathy analysis of FksAp suggests an integral membrane protein with 16 transmembrane helices that coincide with the transmembrane helices of the Saccharomyces Fksps. The sizes of the nontransmembrane domains are strikingly similar to those of Fks1p. The region of FksAp most homologous to the Saccharomyces FKS polypeptides is a large hydrophilic domain of 578 amino acids that is predicted to be cytoplasmic. This domain is 86% identical to the corresponding region of Fks1p and is a good candidate for the location of the catalytic site. Antibodies raised against a peptide derived from the FksAp sequence recognize a protein of ϳ200 kDa in crude membranes and detergent-solubilized active extracts. This protein is enriched ϳ300-fold in GS purified by product entrapment. Purified anti-FksAp immunoglobulin G immunodepletes nearly all of the GS activity in crude or purified extracts when Staphylococcus aureus cells are used to precipitate the antibodies, although it does not inhibit enzymatic activity when added to extracts. The purified GS is inhibited by echinocandins with a sensitivity equal to that displayed by whole cells. Thus, the product of fksA is important for the activity of highly purified preparations of GS, either as the catalytic subunit itself or as an associated copurifying subunit that mediates susceptibility of enzymatic activity to echinocandin inhibition.

Partial purification of a GTP‐insensitive (1 → 3)‐β‐glucan synthase from Phytophthora sojae

“…3). This effect has also been observed with the A. nidulans glucan synthase [18] between 0.7 and 7.1 mM [2,27]. In contrast with other reports, however, both membrane and solubilized preparations displayed no activation of enzyme activity by GTP in the range between 4 gM and 4 raM.…”

“…The fact that the oomycetes have been included in the kingdom Chromista appears to be reflected in these properties that might be called intermediate between plants and fungi. If the 108 kDa protein band that has been primarily enriched by product entrapment represents the glucan synthase for this species, it would also represent a distinctive difference in molecular mass from that reported for yeast and that deduced from homology cloning in a filamentous fungus, such as Aspergillus [18]. Although the inability to dissociate the entrapped protein adequately from the glucan pellet has precluded a measurement of the purification factor, semiquantitative densitometric analysis suggests an enrichment of this band of over two orders of magnitude with this procedure.…”

“…Genes GS-I and KNR4/SMI1 appear to encode transcriptional regulatory proteins [13] while ETGI/FKSI/ CWH53/GSCI and GSC2/FKS2 encode similar 200 kDa proteins which have (1 ~3)-~-glucan synthase activity but are subject to differential regulation during the cell cycle [ 14~ 17]. Similar efforts have been made for medically more relevant genera such as Aspergillus where a FKS1 homolog, JksA, has been isolated which encodes a protein of 200 kDa, subsequently identified and partially purified [18], or Candida where a 187 kDa protein has been enriched [19] and a FKSI/GSCI homolog has been cloned [20]. On the other hand, no [3-(1 ~ 6) linkage forming activity has been identified to date although at least the products of two genes, KRE-6 and SKN1, appear to be involved in (l --* 6)-[3-glucan biosynthesis in yeast [21].…”

Multiple Functional Domains of the Yeast l,3-β-Glucan Synthase Subunit Fks1p Revealed by Quantitative Phenotypic Analysis of Temperature-Sensitive Mutants

“…Only fks1-1093 did not belong to any of these classes. Interestingly, three of four class III mutants (fks1-1114, fks1-1144, and fks1-1154) possessed mutations in the putative catalytic domain (aa 829-973) (Kelly et al 1996).…”

“…The catalytic subunit is Fks1p, a membrane-localized protein (Douglas et al 1994). Fks1p and its alternative protein Fks2p share 88% similarity, including the region of the putative catalytic domain (Mazur et al 1995;Kelly et al 1996). The simultaneous deletion of both genes results in a lethal phenotype, indicating that yeast GS is essential (Inoue et al 1995).…”