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

Abstract

A (1 ~ 3)-~-glucan synthase activity was identified in cell membrane preparations from the oomycete Phytophthora sojae, a soybean pathogen. The activity could be solubilized using the zwitterionic detergent CHAPS at relatively low concentrations (3 mg/ml). High salt concentrations were not effective in removing the activity from the membranes. Detergent solubilization of the enzyme resulted in a six-fold increase of calculated Vma× values (2.5 vs. 0.4 nkat/mg protein) but only minor alteration of the Km (10.6 vs. 10.7 raM). Analysis of the reaction product of the solubilized enzyme by enzymatic degradation and by 2D NMR spectroscopy confirmed its identity as a linear high molecular weight (1 ~ 3)-[~-glucan. Glucan synthase activity in both membrane and solubilized preparations was not activated by GTP or divalent cations as reported for other fungal or plant glucan synthases, The activity was inhibited, as expected, in a competitive manner by UDP with a Ki of 2.9 raM. Partial purification of the enzyme was achieved by anion exchange chromatography followed by product entrapment. This procedure resulted in the selective enrichment of a protein band with apparent Mr 108 000 in SDS-PAGE which was not visible in any of the steps preceding product entrapment. The glucan pellets from product entrapment contained up to 3% of the initial enzyme activity present in the fraction used for the procedure.

Pollen Tubes of Nicotiana alata Express Two Genes from Different β-Glucan Synthase Families

“…A similar phenomenon is observed with the mixedlinkage glucan synthase of corn or barley as, when isolated Golgi membranes are assayed in the presence of UDP-Glc, a (1,3)-␤-glucan synthase activity is detected although no callose is deposited in vivo (Gibeaut and Carpita, 1993;Becker et al, 1995). Oomycetes, filamentous organisms that deposit a wall containing both cellulose and callose, have (1,3)-␤-glucan synthases that are biochemically distinct from those of plants and fungi (Billongrand et al, 1997;Antelo et al, 1998) and that may be a third type of enzyme. Our data also do not bear on the function of the Csl A, B, C, E, and G gene families in plants, and these may encode components of other polysaccharide synthases, such as those responsible for synthesis of the (1,4)-␤-linked backbones of xyloglucan, xylan, or glucomannan.…”

Fungal ß(1,3)-D-glucan synthesis

“…b (1,3)-glucan synthase has been studied in lamentous ascomycetes such as Neurospora crassa [5], Aspergillus nidulans [6], and Aspergillus fumigatus [7,8], in non-lamentous ascomycetes such as Schizosaccharomyces pombe [9], in deuteromycetes such as the dimorphic fungus Candida albicans [10], in basidiomycetes such as Cryptococcus neoformans [11], and even in oomycetes such as Phytophthora spp., which are not true fungi [12,13]. Among these diverse sources, all enzymes are membrane-associated complexes that use UDP-glucose as a substrate (the K M is typically 1-5 mM in crude enzyme preparations) and produce a linear polysaccharide.…”

A putative plant homolog of the yeast β-1,3-glucan synthase subunit FKS1 from cotton (Gossypium hirsutum L.) fibers

“…The disparity between the deduced molecular mass of CFL1p and the position of the band may be attributed to extensive covalent modi®cation of CFL1p during ®ber development such as glycosylation and myristoylation, and possibly protein aggregation during callose synthesis. In addition, callose may tightly associate with the enzyme (Antelo et al 1998), and consequently retard the protein migration in the gel. The pellet labeled by an anti-callose antibody ( Fig.…”