The malate dehydrogenase of Ralstonia eutropha and functionality of the C<subscript>3</subscript>/C<subscript>4</subscript> metabolism in a Tn5-induced mdh mutant

The Tn5-induced mutant VG12 of Ralstonia eutropha HF39, which was isolated in this study, revealed an interesting phenotype: it grew on fructose and pyruvate as well as autotrophically like the wild-type, whereas growth on tricarboxylic acid intermediates and glyoxylic acid was reduced, and no growth occurred if acetate, propionate or levulinate were provided as carbon source. Tn5 was mapped in a gene encoding an NAD(H)-dependent malate dehydrogenase (MDH), and MDH activity was strongly diminished in VG12. Furthermore, the mdh gene was cloned, sequenced and heterologously expressed in Escherichia coli, conferring significantly higher specific MDH activity to the recombinant strain. The phenotype of VG12 sheds light on the C3/C4 metabolism of R. eutropha, which mediates between the Entner–Doudoroff pathway and the tricarboxylic acid cycle (TCC), demonstrating that enzymes catalyzing the conversion of C3 and C4 metabolites can circumvent the metabolic disruption of the TCC in VG12 and that the phosphoenolpyruvate carboxykinase serves a dual and important function. [Copyright &y& Elsevier]