Characterization of two members of a novel malic enzyme class.

The Gram-negative bacterium Rhizobium meliloti contains two distinct malic enzymes. We report the purification of the two isozymes to homogeneity, and their in vitro characterization. Both enzymes exhibit unusually high subunit molecular weights of about 82 kDa. The NAD(P)(+) specific malic enzyme [EC 1.1.1.39] exhibits positive co-operativity with respect to malate, but Michaelis-Menten type behavior with respect to the co-factors NAD(+) or NADP(+). The enzyme is subject to substrate inhibition, and shows allosteric regulation by acetyl-CoA, an effect that has so far only been described for some NADP(+) dependent malic enzymes. Its activity is positively regulated by succinate and fumarate. In contrast to the NAD(P)(+) specific malic enzyme, the NADP(+) dependent malic enzyme [EC 1.1.1.40] shows Michaelis-Menten type behavior with respect to malate and NADP(+). Apart from product inhibition, the enzyme is not subjected to any regulatory mechanism. Neither reductive carboxylation of pyruvate, nor decarboxylation of oxaloacetate, could be detected for either malic enzyme. Our characterization of the two R. meliloti malic enzymes therefore suggests a number of features uncharacteristic for malic enzymes described so far.

Chimeric structure of the NAD(P)+- and NADP+-dependent malic enzymes of Rhizobium (Sinorhizobium) meliloti.

Malic enzymes catalyze the oxidative decarboxylation of malate to pyruvate in conjunction with the reduction of a nicotinamide cofactor. We determined the DNA sequence and transcriptional start sites of the genes encoding the diphosphopyridine nucleotide-dependent malic enzyme (DME, EC 1.1.1.39) and the triphosphopyridine nucleotide-dependent malic enzyme (TME, EC 1.1.1. 40) of Rhizobium (Sinorhizobium) meliloti. The predicted DME and TME proteins contain 770 and 764 amino acids, respectively, and are approximately 320 amino acids larger than previously characterized prokaryotic malic enzymes. The increased size of DME and TME resides in the C-terminal extensions which are similar in sequence to phosphotransacetylase enzymes (EC 2.3.1.8). Modified DME and TME proteins which lack this C-terminal region retain malic enzyme activity but are unable to oligomerize into the native state. Data base searches have revealed that similar chimeric malic enzymes were uniquely present in Gram-negative bacteria. Thus DME and TME appear to be members of a new class of malic enzyme characterized by the presence of a phosphotransacetylase-like domain at the C terminus of the protein.