Glucan synthase of Phytophthora sojae

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). 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. 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. 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. 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.