Pathways of NADPH formation in Escherichia coli.

NADPH formation during growth on glucose was studied in wild type Escherichia coli and in mutants affected, directly or indirectly, in the NADPH-forming reactions glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, malate dehydrogenase, and energy-linked transhydrogenase. The main technique was to grow cells in media containing glucose labeled uniformly with ‘“C and also containing glucose tritiated specifically at positions 1, 3, 4, or 6. Amino acids were isolated from protein and their 3H/*4C ratios determined. For threonine and aspartate, or proline and glutamate, which differ by two NADPH-dependent reductions, one could calculate from the difference in 3H/14C ratios the contribution of hydrogen from each glucose position to the reductions. In some experiments “H/Y ratios were also determined for the fatty acid fraction, and in anaerobic experiments ethanol was isolated. The results were as follows. (a) A strain lacking both glucose-6-phosphate dehydrogenase and isocitrate dehydrogenase still grew on glucose, both aerobically and anaerobically. (b) Unexpectedly, even in a glucose-6-phosphate dehydrogenase mutant or, anaerobically, in an isocitrate dehydrogenase mutant, hydrogens from the 1 and 6 positions of glucose contributed to the biosynthetic reducing pool (NADPH). (c) In the wild type strain, the 3 position hydrogen of glucose also appeared in NADPH; that contribution was lower in anaerobiosis and was absent in a glucose-6phosphate dehydrogenase mutant. (d) In a phosphoglucose isomerase mutant, hydrogens from the 1 and 3 positions of glucose made greater contribution to NADPH than in the wild type strain; a mutant lacking both phosphoglucose isomerase and the membrane ATPase did not grow on glucose. (e) Hydrogen from the 4 position of glucose was a major contributor to NADH, as shown by radioactivity in ethanol. That hydrogen was a minor source of NADPH; the contribution to NADPH was absent in an ATPase mutant CuncB). Accordingly, (a) the hexose monophosphate shunt is probably a minor pathway of NADPH formation in wild type E. coli. (b) The main source of the hydride of NADPH is hydro-