Purification and Characterization of Glucose-6-Phosphate-Dehydrogenase from Cryptococcus neoformans: Identification as "Nothing Dehydrogenase"

Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was purified from Cryptococcus neoformans, a basidiomyceteous yeast that is an opportunistic pathogen of AIDS patients. The enzyme had a subunit molecular weight of 5 × 104, a specific activity of 50 units mg−1, and Km values for NADP and glucose-6-phosphate of 1.6 and 24 μM, respectively. The enzyme catalyzed the dehydrogenation of glucose, in the presence of dimethylsulfoxide, with Km of 5 mM and Vmax 10% of that with glucose-6-phosphate. pH profiles indicated the presence of a group with pKa of 6.6 that is involved in catalysis, and groups with pKas of about 8.8 that are involved in binding of NADP and glucose-6-phosphate. The enzyme was inhibited by NADPH, competitive versus NADP, with Ki of 1 μM, and by zinc ion, competitive versus glucose-6-phosphate, with Ki of 2 μM. Crude enzyme extract catalyzed an appreciable rate of reduction of NADP in the absence of added substrate, a "nothing dehydrogenase" activity. This activity was shown to be due to the presence of glucose-6-phosphate in the crude extract. It was calculated that cells of C. neoformans contain about 25 μmol of glucose-6-phosphate per gram, wet weight.