Nudix hydrolase controls nucleotides and glycolytic mechanisms in hypoxic Aspergillus nidulans

Nucleoside diphosphates linked to moiety X (Nudix) hydrolase functions were investigated in hypoxic Aspergillus nidulans cells. Among three nudix hydrolase isozymes, NdxA transcription was up-regulated under oxygen (O2)-limited conditions. A gene disruptant of the NdxA-encoding gene (NdxAΔ) accumulated more NADH and ADP-ribose than the wild type (WT) under the same conditions. These results indicate that NdxA hydrolyzes these nucleotides in hypoxic fungal cells, which accords with the thesis that NdxA hydrolyzes NADH and ADP-ribose. Under O2-limited conditions, NdxAΔ decreased glucose consumption, the production of ethanol and lactate, cellular ATP levels, and growth as compared with WT. WT cultured under hypoxia converted exogenously added fructose 1,6-bisphophate, a glycolytic intermediate, to glyceraldehyde 3-phosphate (GAP). The hypoxic NdxAΔ cells accumulated 3.0- to 4.2-fold more GAP than WT under the same conditions, indicating that NdxA increased GAP oxidation by a glycolytic mechanism. Steady-state kinetics indicated that NADH and ADP-ribose competitively inhibited fungal GAP dehydrogenase (GAPDH) with Ki values of 34- and 55-µM, respectively. These results indicate that NdxA hydrolyzes cellular NADH- and ADP-ribose, derepresses GAPDH activity, and hence up-regulates glycolysis in hypoxic A. nidulans cells. That NdxAΔ consumed less pyruvate and tricarboxylate cycle intermediates than WT suggests that NdxA-dependent hydrolysis of nucleotides controls the catabolism of these carbon sources under O2-limited conditions.