Biochemical and genetic characterization of the three metabolic routes in Thermococcus kodakarensis linking glyceraldehyde 3‐phosphate and 3‐phosphoglycerate

Summary In the classical Embden–Meyerhof (EM) pathway for glycolysis, the conversion between glyceraldehyde 3‐phosphate (GAP) and 3‐phosphoglycerate (3‐PGA) is reversibly catalysed by phosphorylating GAP dehydrogenase (GAPDH) and phosphoglycerate kinase (PGK). In the Euryarchaeota Thermococcus kodakarensis and Pyrococcus furiosus, an additional gene encoding GAP:ferredoxin oxidoreductase (GAPOR) and a gene similar to non‐phosphorylating GAP dehydrogenase (GAPN) are present. In order to determine the physiological roles of the three routes that link GAP and 3‐PGA, we individually disrupted the GAPOR, GAPN, GAPDH and PGK genes (gor, gapN, gapDH and pgk respectively) of T. kodakarensis. The Δgor strain displayed no growth under glycolytic conditions, confirming its proposed function to generate reduced ferredoxin for energy generation in glycolysis. Surprisingly, ΔgapN cells also did not grow under glycolytic conditions, suggesting that GAPN plays a key role in providing NADPH under these conditions. Disruption of gor and gapN had no effect on gluconeogenic growth. Growth experiments with the ΔgapDH and Δpgk strains indicated that, unlike their counterparts in the classical EM pathway, GAPDH/PGK play a major role only in gluconeogenesis. Biochemical analyses indicated that T. kodakarensis GAPN did not recognize aldehyde substrates other than d‐GAP, preferred NADP+ as cofactor and was dramatically activated with glucose 1‐phosphate.