Gerhard Loeber

Crystal structure of human mitochondrial NAD(P)+-dependent malic enzyme: a new class of oxidative decarboxylases.

Background: Malic enzymes catalyze the oxidative decarboxylation of malate to pyruvate and CO(2) with the concomitant reduction of NAD(P)(+) to NAD(P)H. They are widely distributed in nature and have important biological functions. Human mitochondrial NAD(P)(+)-dependent malic enzyme (mNAD-ME) may have a crucial role in the metabolism of glutamine for energy production in rapidly dividing cells and tumors. Moreover, this isoform is unique among malic enzymes in that it is a cooperative enzyme, and its activity is controlled allosterically. Results: The crystal structure of human mNAD-ME has been determined at 2.5 Å resolution by the selenomethionyl multiwavelength anomalous diffraction method and refined to 2.1 Å resolution. The structure of the monomer can be divided into four domains; the active site of the enzyme is located in a deep cleft at the interface between three of the domains. Three acidic residues (Glu255, Asp256 and Asp279) were identified as ligands for the divalent cation that is required for catalysis by malic enzymes. Conclusions: The structure reveals that malic enzymes belong to a new class of oxidative decarboxylases. The tetramer of the enzyme appears to be a dimer of dimers. The active site of each monomer is located far from the tetramer interface. The structure also shows the binding of a second NAD(+) molecule in a pocket 35 Å away from the active site. The natural ligand for this second binding site may be ATP, an allosteric inhibitor of the enzyme.

Structure of a closed form of human malic enzyme and implications for catalytic mechanism.

Malic enzymes are widely distributed in nature and have many biological functions. The crystal structure of human mitochondrial NAD(P)+-dependent malic enzyme in a quaternary complex with NAD+, Mn++ and oxalate has been determined at 2.2 Å resolution. The structures of the quaternary complex with NAD+, Mg++, tartronate or ketomalonate have been determined at 2.6 Å resolution. The structures show the enzyme in a closed form in these complexes and reveal the binding modes of the cation and the inhibitors. The divalent cation is coordinated in an octahedral fashion by six ligating oxygens, two from the substrate/inhibitor, three from Glu 255, Asp 256 and Asp 279 of the enzyme, and one from a water molecule. The structural information has significant implications for the catalytic mechanism of malic enzymes and identifies Tyr 112 and Lys 183 as possible catalytic residues. Changes in tetramer organization of the enzyme are also observed in these complexes, which might be relevant for its cooperative behavior and allosteric control.

The use of Genetically Engineered Cells in Drug Discovery

Modern drug discovery is changing at a very rapid pace. Techniques directed at rational drug design employing structural biology [for review, see (1)], computer aided molecular modeling and screening (2, 3) and the use of expert systems and neural networks (4) are being integrated in the drug discovery process of pharmaceutical companies. In contrast, the classical approach to drug discovery, rand om screening of isolated chemical compounds or natural extracts, has been employed successfully for the last decades. Technical advances during the last 5 years in laboratory automation, miniaturization and data processing have made it possible to test 10, 000 single compounds per day and more. Major efforts are made to test entire compound collections of large pharmaceutical companies which can consist of more than 500, 000 substances within weeks. The advances in molecular biology have added to the drug discovery processes in several aspects. The understand ing of critical processes in signaling and in gene expression identified key components of cellular regulation leading to the identification of targets as drug intervention sites in pathological processes. Advances in genetic engineering also provided valuable tools to design cellular assay systems to study target regulatory molecules such as receptors or other enzymes in their normal environment and coupled to their correct signaling cascades. For this purpose, the desired target molecule has to be expressed in the respective cell and the downstream signaling has to be linked to an appropriate reporter system. Possible readout systems include luminescence, fluorescence, time resolved fluorescence, uptake or release of radioactivity, or colorimetric techniques.