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

Abstract

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.

Identification of Domains Involved in Tetramerization and Malate Inhibition of Maize C4-NADP-Malic Enzyme

“…1 Fellows of the Consejo Nacional de Investigaciones Científicas y Té cnicas. 2 Members of the Researcher Career of the Consejo Nacional de Investigaciones Científicas y Té cnicas. 3 To whom correspondence should be addressed.…”

“…The amino acid sequences of malic enzymes are highly conserved among many organisms, from bacteria to human. However, malic enzymes do not show recognizable amino acid sequence homology to other proteins such as dehydrogenases, decarboxylases, or other oxidative decarboxylases, comprising a new class of oxidative decarboxylases (2).…”

“…The photosynthetic plastidic isoform, which is expressed in bundle sheath cells of some C 4 plants, represents a unique and specialized form of NADP-ME with particular kinetic, structural, and regulatory properties, and all of these make this enzyme suitable to participate in the CO 2 concentrating mechanism that increases the photosynthetic yield of NADP-ME C 4 plants (3,4). In contrast, C 4 nonphotosynthetic NADP-ME isoforms are expressed in the cytosol (5) and plastids (6 -9).…”

Functional Roles of the Tetramer Organization of Malic Enzyme

“…Enzymes-The expression and purification protocols for human m-NAD(P)-ME have been described in previous reports (4,6). Briefly, m-NAD(P)-ME was subcloned into the expression vector (pRH281) and transformed into Escherichia coli BL21 cells for overexpression using the inducible trp promoter system.…”

“…Malic enzymes are found in a broad spectrum of living organisms that share conserved amino acid sequences and structural topology; such shared characteristics reveal a crucial role for the biological functions of these enzymes (4,5). In mammals, malic enzymes have been divided into three isoforms according to their cofactor specificity and subcellular localization as follows: cytosolic NADP ϩ -dependent (c-NADP-ME), 2 mitochondrial NADP ϩ -dependent (m-NADP-ME), and mitochondrial NAD(P) ϩ -dependent (m-NAD(P)-ME).…”