Functions of the Membrane-Associated and Cytoplasmic Malate Dehydrogenases in the Citric Acid Cycle of Escherichia coli

Abstract

ABSTRACT Oxidation of malate to oxaloacetate in Escherichia coli can be catalyzed by two enzymes: the well-known NAD-dependent malate dehydrogenase (MDH; EC 1.1.1.37 ) and the membrane-associated malate:quinone-oxidoreductase (MQO; EC 1.1.99.16 ), encoded by the gene mqo (previously called yojH ). Expression of the mqo gene and, consequently, MQO activity are regulated by carbon and energy source for growth. In batch cultures, MQO activity was highest during exponential growth and decreased sharply after onset of the stationary phase. Experiments with the β-galactosidase reporter fused to the promoter of the mqo gene indicate that its transcription is regulated by the ArcA-ArcB two-component system. In contrast to earlier reports, MDH did not repress mqo expression. On the contrary, MQO and MDH are active at the same time in E. coli . For Corynebacterium glutamicum , it was found that MQO is the principal enzyme catalyzing the oxidation of malate to oxaloacetate. These observations justified a reinvestigation of the roles of MDH and MQO in the citric acid cycle of E. coli . In this organism, a defined deletion of the mdh gene led to severely decreased rates of growth on several substrates. Deletion of the mqo gene did not produce a distinguishable effect on the growth rate, nor did it affect the fitness of the organism in competition with the wild type. To investigate whether in an mqo mutant the conversion of malate to oxaloacetate could have been taken over by a bypass route via malic enzyme, phosphoenolpyruvate synthase, and phosphenolpyruvate carboxylase, deletion mutants of the malic enzyme genes sfcA and b2463 (coding for EC 1.1.1.38 and EC 1.1.1.40 , respectively) and of the phosphoenolpyruvate synthase (EC 2.7.9.2 ) gene pps were created. They were introduced separately or together with the deletion of mqo . These studies did not reveal a significant role for MQO in malate oxidation in wild-type E. coli . However, comparing growth of the mdh single mutant to that of the double mutant containing mdh and mqo deletions did indicate that MQO partly takes over the function of MDH in an mdh mutant.