Structural studies of the pigeon cytosolic NADP(+)-dependent malic enzyme

Abstract

Malic enzymes are widely distributed in nature, and have important biological functions. They catalyze the oxidative decarboxylation of malate to produce pyruvate and CO(2) in the presence of divalent cations (Mg(2+), Mn(2+)). Most malic enzymes have a clear selectivity for the dinucleotide cofactor, being able to use either NAD(+) or NADP(+), but not both. Structural studies of the human mitochondrial NAD(+)-dependent malic enzyme established that malic enzymes belong to a new class of oxidative decarboxylases. Here we report the crystal structure of the pigeon cytosolic NADP(+)-dependent malic enzyme, in a closed form, in a quaternary complex with NADP(+), Mn(2+), and oxalate. This represents the first structural information on an NADP(+)-dependent malic enzyme. Despite the sequence conservation, there are large differences in several regions of the pigeon enzyme structure compared to the human enzyme. One region of such differences is at the binding site for the 2'-phosphate group of the NADP(+) cofactor, which helps define the cofactor selectivity of the enzymes. Specifically, the structural information suggests Lys362 may have an important role in the NADP(+) selectivity of the pigeon enzyme, confirming our earlier kinetic observations on the K362A mutant. Our structural studies also revealed differences in the organization of the tetramer between the pigeon and the human enzymes, although the pigeon enzyme still obeys 222 symmetry.

Fig. 1.

Crystal structure of pigeon cytosolic NADP⁺-dependent malic enzyme. (A) Schematic drawing of the structure of pigeon c-NADP-ME in complex with NADP⁺, oxalate, and Mn²⁺. The β strands are shown in cyan, α helices in yellow, and the connecting loops in purple. NADP⁺ and oxalate are shown as stick models, and Mn²⁺ is shown as a purple sphere. The NAD⁺ molecule in the second site is present only in the human ME structure (Xu et al. 1999). (B) Structure comparison between human m-NAD-ME (shown in cyan) and pigeon c-NADP-ME (in yellow). The two regions where there are deletions in the pigeon enzyme are also indicated. (C) Plot of the distances between equivalent Cα atoms of the pigeon and human ME structures. (D) Electron density for the NADP⁺ molecule after noncrystallographic symmetry averaging over the four tetramers. The refined atomic model is shown for reference. (A) and (B) were created with Ribbons (Carson 1987), and (D) was created with SETOR (Evans 1993).

Fig. 2.

The active site of the pigeon malic enzyme. (A) Stereo drawing showing the active site of pigeon c-NADP-ME. The oxalate molecule is shown in green for carbon atoms, NADP⁺ in green, and protein residues in gray. The Mn²⁺ cation is shown as a purple sphere. (B) Molecular surface of pigeon c-NADP-ME near the active site region, colored according to electrostatic potential. The NADP⁺ molecule is shown as a stick model. (C) Comparison of the binding modes of NADP⁺ to the pigeon ME (in green for carbon atoms) and of NAD⁺ to the human ME (in cyan). The oxalate and the Mn²⁺ ion are also shown in the comparison. (A) was created with Ribbons (Carson 1987), and (B) and (C) were created with Grasp (Nicholls et al. 1991).

Fig. 3.

A possible molecular mechanism for cofactor selectivity. (A) Stereo drawing showing the structure comparison between human m-NAD-ME (in cyan for carbon atoms) and pigeon c-NADP-ME (in green) near the 2`-phsophate of NADP<sup>+</sup>. (B) Alignment of ME sequences near the binding site for the 2`-phosphate group of NADP⁺. The cofactor dependence of the various malic enzymes is indicated. The Asp345:Arg354 ion-pair is shown in purple. (A) was created with Grasp (Nicholls et al. 1991).

Fig. 4.

The tetramer of the pigeon malic enzyme. (A) Schematic drawing showing the tetramer of the pigeon ME. The four monomers are given different colors. The dimer and the tetramer interfaces are labeled. (B) The interactions of the C-terminal tail in one molecule (shown as stick models in cyan) with the other dimer of the tetramer (shown as molecular surfaces colored green and yellow, with their C termini labeled with the letter "C"). (C–E). Detailed structure comparisons of residues 541–546, in the tetramer interface, between the pigeon (C) and the closed (D) and open (E) forms of the human malic enzymes. These residues are located in the tetramer interface, indicated by the red oval in (A). The twofold axis is indicated with the purple oval. (A) was created with Ribbons (Carson 1987), (B–E) were created with Grasp (Nicholls et al. 1991).