Biosynthesis of UDP-GlcA, a key metabolite for capsular polysaccharide synthesis in the pathogenic fungus Cryptococcus neoformans

UDP-glucose dehydrogenase catalyses the conversion of UDP-glucose into UDP-GlcA, a critical precursor for glycan synthesis across evolution. We have cloned the gene encoding this impor-tant enzyme from the opportunistic pathogen Cryptococcus neo-formans. In this fungus, UDP-GlcA is required for the synthesis of capsule polysaccharides, which in turn are essential for virulence. The gene was expressed in Escherichia coli and the 51.3-kDa re-combinant protein from wild-type and five mutants was purified for analysis. The cryptococcal enzyme is strongly inhibited by UDP-xylose and NADH, has highest activity at pH 7.5 and de-monstrates Km (app) values of 0.1 and 1.5 mM for NAD+ and UDP-glucose respectively. Its activity was significantly decreased by mutations in the putative sites of NAD+ and UDP-glucose bind-ing. Unlike previously reported eukaryotic UDP-glucose dehydro-genases, which are hexamers, the cryptococcal enzyme is a dimer. Key words: capsule, Cryptococcus neoformans, pathogenic fungi, UDP-GlcA, UDP-glucose dehydrogenase