The Role of (1,3)-β-D-Glucan Synthase in Fungal Cell Wall Biosynthesis

(1,3)-β-D-glucan synthase is responsible for the biosynthesis of (1,3)-β-D-glucan, a

major component of the fungal cell wall, and is a proven target of clinically approved

antifungal antibiotics. Due to emerging drug resistance and the limited oral availabil-

ity of existing drugs, there is growing interest in developing existing (1,3)-β-D-glucan

synthase inhibitors as well as discovering new ones. However, development has been

hampered by a limited understanding of the mechanisms of both the catalysis and

inhibition of (1,3)-β-D-glucan synthase. This dissertation is an attempt to increase

our understanding of (1,3)-β-D-glucan synthase catalysis and its role in fungal cell

wall biosynthesis.

I describe the development of a size exclusion chromatography-based method

that allowed characterization of the lengths and amounts of (1,3)-β-D-glucan formed

in assays, allowing the first functional and mechanistic characterization of (1,3)-β-

D-glucan synthase. I also detail the synthesis and use of substrate analogs that

provided strong support that (1,3)-β-D-glucan synthase elongates (1,3)-β-D-glucan

chains from the non-reducing end, and does so processively with a turnover of ~0.5

(1,3)-β-D-glucan min -1 . Based on the results, I propose a model for the catalytic

mechanism of (1,3)-β-D-glucan synthase. The observation that glucanosyltransferase

Gas1p is unnecessary for formation of (1,3)-β-D-glucan with biologically relevant

degree of polymerization supports the model of fungal cell wall biosynthesis where

(1,3)-β-D-glucan synthase is sufficient for generating (1,3)-β-D-glucan.

ivAdditionally, I provide evidence that (1,3)-β-D-glucan synthase fractionates into

detergent resistant membranes, and provide several strategies to overcome the chal-

lenge of purifying (1,3)-β-D-glucan synthase for further biochemical studies. The

techniques developed and reported here can also be applied to characterize other

polysaccharide synthases.