Length Specificity and Polymerization Mechanism of (1,3)-β‑d‑Glucan Synthase in Fungal Cell Wall Biosynthesis

(1,3)-β-d-Glucan synthase (GS) catalyzes formation
of the linear (1,3)-β-d-glucan in the fungal cell wall
and is a target of clinically approved antifungal antibiotics. The
catalytic subunit of GS, FKS protein, does not exhibit significant
sequence homology to other glycosyltransferases, and thus, significant
ambiguity about its catalytic mechanism remains. One of the major
technical barriers in studying GS is the absence of activity assay
methods that allow characterization of the lengths and amounts of
(1,3)-β-d-glucan due to its poor solubility in water
and organic solvents. Here, we report a successful development of
a novel GS activity assay based on size-exclusion chromatography coupled
with pulsed amperometric detection and radiation counting (SEC-PAD-RC),
which allows for the simultaneous characterization of the amount and
length of the polymer product. The assay revealed that the purified
yeast GS produces glucan with a length of 6550 ± 760 mer, consistent
with the reported degree of polymerization of (1,3)-β-d-glucan isolated from intact cells. Pre-steady state kinetic analysis
revealed a highly efficient but rate-determining chain elongation
rate of 51.5 ± 9.8 s–1, which represents the
first observation of chain elongation by a nucleotide–sugar-dependent
polysaccharide synthase. Coupling the SEC-PAD-RC method with substrate
analogue mechanistic probes provided the first unambiguous evidence
that GS catalyzes non-reducing end polymerization. On the basis of
these observations, we propose a detailed model for the catalytic
mechanism of GS. The approaches described here can be used to determine
the mechanism of catalysis of other polysaccharide synthases.