Characterization of the need for galactofuranose during the Neurospora crassa life cycle.

Galactofuranose is a constituent of the cell walls of filamentous fungi. The galactofuranose can be found as a component of N-linked oligosaccharides, in O-linked oligosaccharides, in GPI-anchored galactomannan, and in free galactomannan. The Neurospora genome contains a single UDP-galactose mutase gene (ugm-1/NCU01824) and two UDP-galactofuranose translocases used to import UDP-galactofuranose into the lumen of the Golgi apparatus (ugt-1/NCU01826 and ugt-2/NCU01456). Our results demonstrate that loss of galactofuranose synthesis or its translocation into the lumen of the secretory pathway affects the morphology and growth rate of the vegetative hyphae, the production of conidia (asexual spores), and dramatically affects the sexual stages of the life cycle. In mutants that are unable to make galactofuranose or transport it into the lumen of the Golgi apparatus, ascospore development is aborted soon after fertilization and perithecium maturation is aborted prior to the formation of the neck and ostiole. The Neurospora genome contains three genes encoding possible galactofuranosyltransferases from the GT31 family of glycosyltransferases (gfs-1/NCU05878, gfs-2/NCU07762, and gfs-3/NCU02213) which might be involved in generating galactofuranose-containing oligosaccharide structures. Analysis of triple KO mutants in GT31 glycosyltransferases shows that these mutants have normal morphology, suggesting that these genes do not encode vital galactofuranosyltransferases.

Cloning, characterization and expression of a gene encoding endo-1, 4- ß-xylanase from the fungus Termitomyces clypeatus.

Enzymatic degradation of hemi-cellulosic substrates has gained plenty of industrial attentions recently. Complete enzymatic degradation of complex and recalcitrant hemicellulose requires an enzymatic cocktail consisting primarily of endo-1,4-ß-xylanase (xyl), ß-xylosidase, arabinofuranosidase etc. This article reports, for the first time, the identification, cloning, expression and partial characterization of a potent endo-1,4- ß-xylanase gene (pxyl) from the mushroom Termitomyces clypeatus (TC) in E. coli and S. cerevisiae. The cDNA for pxyl was found to be 678 bp that in turn gives rise to a precursor protein (Pxyl) of 225 amino acids long when cloned in prokaryotic expression vector. To characterize additionally, the cDNA was also expressed in S. cerevisiae. Bioinformatics study predicted that the Pxyl contains a 19 amino acid long leader peptide that enables post translational modifications including glycosylation as well as its efficient secretion in the medium. The recombinant protein has been found to be a member of GH11 family containing two distant glutamic acids as catalytic residues. This report describes yet another new and potent source of xylanase for commercial exploitation by industry in future.

Putative role of invariant water molecules in the X-ray structures of family G fungal endoxylanases.

Fungal endo-1,4-beta-xylanases (EC3.2.1.8), because of their widespread industrial applications have become one of the most researched industrial enzymes in recent times. Despite its significance, the role of conserved water molecules in the catalytic activities and structural stability of these enzymes from the fungi have not been studied to a great extent. Our computational structural bioinformatics and MD simulation studies have identified the existence of seven invariant water molecules (IW1- IW7) and reveals the stereo-chemical and electronic consequences of those conserved water molecules in G-xylanase enzyme from eight different fungi. The buried water molecules IW1 and IW2 may have decisive roles in catalysis and may also be associated with ligand binding process of the enzyme, whereas IW3, IW4, IW5, IW6 and IW7 may be involved in stabilizing the important (H144/R145) residues through H-bonds. Possibly they are also involved in the stabilization of secondary structures and anchor to maintain its stereo-chemical architecture. Moreover, a distorted 'W' shaped signature geometry that is observed at the surface of the enzyme can be used to identify the hydrophilic centers in the electron density map of other unknown members of the family G xylanases. The results from this computational investigation could be of interest to a large number of researchers working with the xylanases.

Molecular structure and catalytic mechanism of fungal family G acidophilic xylanases.

Industrial applications of xylanases have made this enzyme an important subject of applied research work. Function of this particular enzyme is to degrade or hydrolyze the plentiful polysaccharide xylan, an important component of hemicellulose. It mainly cleaves the backbone of xylan that is made up of a number of xylose residues connected with ß-1,4-glycosidic linkages. Fungi with mycelia are regarded as the best producer of xylanases. These varied xylanases not only differ in their sizes and shapes but also differ in their physicochemical properties. Depending on the optimum pH in which they work best, they have been classified into (1) acidophilic xylanases active at low pH or acidic pH range, (2) alkaliphilic xylanases that are active at high or alkaline pH range and (3) neutral xylanases having pH optima in the neutral range between pH 5 and 7. Other researchers have classified the xylanases also on the basis of their structural properties, kinetic parameters, etc. This review discusses the molecular structures of some acidophilic xylanases and the molecular basis of low pH optima observed for their activities. It also discusses their unique catalytic mechanism and actual role of the catalytic residues found in them. Apart from these, the review also discusses different applications of these acidophilic xylanases in different industries. The article concludes with brief suggestions about how these acidophilic xylanases can be created employing the techniques of genetic engineering and concepts of synthetic evolution, using the traits of the known acidophilic xylanases discussed in the review.