Cloning and Expression of Pigeon Liver Cytosolic NADP+-Dependent Malic Enzyme cDNA and Some of Its Abortive Mutants

Abstract

A full-length 1927-base-pair cDNA of pigeon liver malic enzyme was obtained by utilizing the screening of the cDNA library and polymerase chain reaction techniques. The cDNA contained one open reading frame coding for 557 amino acid residues, flanked by 86 and 167 nucleotides at the 5′ and 3′ termini, respectively, and was successfully cloned and expressed in Escherichia coli cells. Functionally active recombinant malic enzyme was purified from the cells. This recombinant enzyme has a Km value for L-malate of 160 ± 30 μM, which is almost identical to that for the natural enzyme (150 ± 17 μM). The Km value for Mn2+ (4.2 ± 0.3 μM) is higher than that for the natural pigeon malic enzyme (1.4 ± 0.2 μM), while the Km value for NADP+ (3.8 ± 0.3 μM) is lower than that for the natural enzyme (10.8 ± 0.1 μm). The catalytic constant (kcat) for the recombinant enzyme is decreased by 3.6-fold, but the substrate inhibition constant for L-malate is increased by about 40-fold. Change in the quaternary structure of the recombinant enzyme was revealed in the pH perturbation examination. A truncated pigeon liver malic enzyme, lacking the first 13 amino acid residues, and a recombinant protein, mutated at F19S, N250S, and L353Q, showed no enzymatic activity. Both abortive recombinant mutant proteins were still able to bind with 2′,5′-ADP agarose; however, the fluorescence emission spectrum of the protein bound NADPH did not show a blue shift as the natural enzyme. In accordance with these observations, we suggest that the adenosine 2′,5′-bisphosphate binding domain of the NADP+ binding site in the βαβ motif may still be retained in these mutant proteins. However, the local hydrophobic environment for the binding of the nicotinamide moiety of the coenzyme molecule may be altered. Therefore, the lack of catalytic activity of the mutant proteins could be attributed to an improper orientation of the bound NADP+.