Creation of an NADP-dependent pyruvate dehydrogenase multienzyme complex by protein engineering

The systematic conversion of the NAD-dependent enzyme, Escherichia coli dihydrolipoamide dehydrogenase, into one that rejects NAD but is fully active with NADP is presented. In addition, the ability of the mutated enzyme to assemble into a fully active pyruvated dyhydrogenase multicomplex enzyme complex was examined. The specificity of the assembled product was also assessed. It was found to catalyze the oxidative decarboxylation of pyruvate to yield acetyl coenzyme-A.