Regulation of NAD⁺‐ and NADP⁺‐linked isocitrate dehydrogenase in the obligate methylotrophic bacterium Pseudomonas W6

Abstract

Abstract Cell‐free extracts of the obligate methanol‐utilizing bacterium Pseudomonas W6 catalyze the oxydation of isocitrate to α‐ketoglutarate in the presence of NAD + and NADP + . After electro‐focusing of the crude extract of Pseudomonas W6 actually two distinct bands each of NAD + ‐linked isocitrate dehydrogenase (NAD + ‐IDH) and of NADP + ‐linked isocitrate dehydrogenase (NADP + ‐IDH) could be observed. The NAD + ‐IDH was completely separated from the NADP + ‐IDH by employing DEAE ion exchange chromatography and further purified by affinity chromatography using Cibacron blue F 3G‐A. The NAD + ‐IDH was inhibited by a high energy charge, whereas the NADP + ‐IDH was found to be independent of energy charge. Consequently the NAD + ‐IDH showed the control behaviour of an enzyme of an energy‐generating sequence which, however, equally fulfils a catabolic and an anabolic function. With respect to the inhibition by reduced pyridine nucleotides and α‐ketoglutarate differences between NAD + ‐IDH and NADP + ‐IDH were also found. Only the NADP + ‐linked enzyme exhibited a feedback inhibition by its reaction products α‐ketoglutarate and NADPH. This control behaviour gives evidence for the biosynthetic function of the NADP + ‐IDH. These results confer an amphibolic character to the sequence from citrate to α‐ketoglutarate in the incomplete citric‐acid cycle of Pseudomonas W6.