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 important enzyme from the opportunistic pathogen Cryptococcus neoformans. 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 recombinant 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 demonstrates 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 binding. Unlike previously reported eukaryotic UDP-glucose dehydrogenases, which are hexamers, the cryptococcal enzyme is a dimer.
Bar-Peled
Maor
M
Complex Carbohydrate Research Center of the University of Georgia, 220 Riverbend Road, Athens, GA 30602-4712, USA.
Griffith
Cara L
CL
Ory
Jeramia J
JJ
Doering
Tamara L
TL
D000595:Amino Acid Sequence; D029681:Arabidopsis Proteins / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry; D003001:Cloning, Molecular / Q000379:methods; D017124:Conserved Sequence / Q000235:genetics; D003455:Cryptococcus neoformans / Q000201:enzymology* / Q000472:pathogenicity; D018076:DNA, Complementary / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000096:biosynthesis* / Q000235:genetics; D016415:Sequence Alignment / Q000379:methods; D014533:Uridine Diphosphate Glucose Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014535:Uridine Diphosphate Glucuronic Acid / Q000096:biosynthesis*
D029681:Arabidopsis Proteins; D001426:Bacterial Proteins; D018076:DNA, Complementary; D004271:DNA, Fungal; D005656:Fungal Proteins; D011134:Polysaccharides; D014535:Uridine Diphosphate Glucuronic Acid; D014533:Uridine Diphosphate Glucose Dehydrogenase