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1 Department of Biological Sciences, Columbia University, New York, New York 10027, USA
2 Department of Biochemistry, National Defense Medical Center, Taipei 114, Taiwan
3 Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan
Reprint requests to: Liang Tong, Department of Biological Sciences, Columbia University, New York, NY 10027; e-mail: tong{at}como.bio.columbia.edu; fax: (212) 854-5207.
Malic enzymes are widely distributed in nature, and have important biological functions. They catalyze the oxidative decarboxylation of malate to produce pyruvate and CO2 in the presence of divalent cations (Mg2+, Mn2+). 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+, Mn2+, 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.
Keywords: Malic enzyme; oxidative decarboxylase; cofactor selectivity; protein structure
Abbreviations: CCD, charge-coupled device • c-NADP-ME, cytosolic NADP+-dependent malic enzyme • DTT, dithiothreitol • IPTG, isopropyl ß-D-thio-galactopyranoside • ME, malic enzyme • m-NAD-ME, mitochondrial NAD+-dependent malic enzyme • NCS, noncrystallographic symmetry • NSLS, national synchrotron light source • OD, optical density • PEG, polyethylene glycol • PMSF, phenylmethylsulfonyl fluoride • RMS, root mean square
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