Glucan synthase of Phytophthora sojae
b'Glucans, with the (1-3)-b-glucosidic linkage as major feature, are present in most of the higher plants, in many lower plants, as well as in microorganisms (Stone and Clarke, 1992). The synthesis of (1-3)-b-glucan in vivo is catalysed by the enzyme (1-3)-b-glucan synthase (EC 2.4.1.34; UDP-glucose:1,3-b-D-glucan 3-b-D-glucosyl transferase) using UDP-glucose as substrate. The (1-3)-b-glucan synthase was characterised in a number of fungi and plants, but not much work was done with oomycetes (Stone and Clarke, 1992), even though one of the earliest successful in vitro assays for glucan synthase activity was done using Phytophthora cinnamomi (Wang and Bartnicki-Garcia, 1976, Selitrennikoff 1995).\\n In this work, the glucan synthase of the oomycete Phytophthora sojae was characterised, solubilized, and partially purified, and the cDNA for a protein co-purifying with the glucan synthase activity was cloned.\\n The glucan synthase of P. sojae had several features that distinguish it from what is known for glucan synthases from fungi and plants (callose synthases). Its apparent Km value for UDP-glucose was higher than reported for other glucan synthases. The activity was GTP-independent and shown not to be activated by divalent cations like Mg2+ or Ca2+, and shown to be inhibited by some others, like Cu2+ or Zn2+. Some of these properties are shared with the glucan synthase from Achlya ambisexualis (Cabib and Kang, 1987), an organism that belongs to the same kingdom as P. sojae: the Chromista. \\n It was also demonstrated by NMR analysis and enzymatic degradation that the sole product of the CHAPS-solubilized glucan synthase of P. sojae was composed of long linear (1-3)-b-glucan chains.\\n The glucan synthase was purified by product entrapment. Two proteins, with apparent molecular masses of 108 and 50 kDa, were enriched and microsequenced. With the degenerated oligonucleotides derived from the sequenced peptides, PCR experiments were performed using as a template a cDNA library of actively growing P. sojae mycelium. No positive result could be obtained by using the oligonucleotides derived from the 108 kDa protein. In contrast, a full length cDNA (named Ps-P50) was cloned, using the oligonucleotides derived from the 50 kDa protein (P50). The deduced amino acid sequence of Ps-P50 cDNA contains sequence motifs homologous to the peptides sequenced from P50. This cDNA encodes a protein with a molecular mass of 49.991 Da with no homology found in the data bases. Diversity between the PCR product and the cDNA clone, and various different homologous ESTs indicates that Ps-P50 is a member of a gene family.'