Crystallographic Studies on Ascaris suum NAD-Malic Enzyme Bound to Reduced Cofactor and Identification of an Effector Site

Rao, G. Gopala; Coleman, David E.; Karsten, William E.; Cook, Paul F.; Harris, Ben G.

doi:10.1074/jbc.m305145200

Cited by 32 publications

(44 citation statements)

References 33 publications

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“…These structural data establish malic enzymes as a new class of oxidative decarboxylases with a discrete backbone structure (6,8,10,31). Human m-NAD-ME in various complexes with malate/ pyruvate, Mn 2ϩ /Mg 2ϩ , NAD ϩ , fumarate, and transition state analog inhibitors has been reported (6,7,(32)(33)(34).…”

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confidence: 79%

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Determinants of the Dual Cofactor Specificity and Substrate Cooperativity of the Human Mitochondrial NAD(P)+-dependent Malic Enzyme

Hsieh

Liu

Chang³

et al. 2006

Journal of Biological Chemistry

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The human mitochondrial NAD(P)؉ -dependent malic enzyme (m-NAD-ME) is a malic enzyme isoform with dual cofactor specificity and substrate binding cooperativity. to Lys in human m-NAD-ME changes it to an NADP ؉ -dependent enzyme, which is characteristic because it is non-allosteric, non-cooperative, and NADP ؉ -specific.Malic enzymes are a newly discovered family of oxidative decarboxylases that catalyze the conversion of the substrate L-malate to CO 2 and pyruvate, with concomitant reduction of NAD(P) ϩ to NAD(P)H (1-5). Divalent metal ion (Mn 2ϩ or Mg 2ϩ ) is required for the enzyme-catalyzed reaction. These enzymes exist ubiquitously in nature, with conserved sequences and similar overall structural topology among different species (6 -10). In mammalian cells, according to their cofactor specificity, three isoforms of malic enzyme have been recognized: cytosolic NADP ϩ -dependent (c-NADP-ME) 3 (11, 12), mitochondrial NADP ϩ -dependent (13), and mitochondrial NAD(P) ϩ -dependent (m-NAD-ME) (3, 14). The third isoform, m-NAD-ME, can utilize both NAD ϩ and NADP ϩ as a cofactor, but it prefers NAD ϩ under physiological conditions (3). Via the NADH and pyruvate products, human m-NAD-ME may play an important role in the metabolism of glutamine in fast growing tissues and tumors (3, 14 -21).Distinctive from the other two mammalian isoforms, m-NAD-ME has a complex regulatory system that controls its catalytic activity (22)(23)(24). It displays cooperative behavior with respect to the substrate L-malate, and the enzyme activity can be allosterically activated by fumarate (19,(22)(23)(24)(25)(26)(27)(28). Furthermore, previous studies have suggested that the inhibitory effect of ATP may operate through an allosteric mechanism (19,22,26,29,30), and the allosteric properties of m-NAD-ME imply its specific role in the pathways of malate and glutamine oxidation in tumor mitochondria (18 -22, 25). However, site-directed mutagenesis and kinetic studies have demonstrated that ATP may actually act as an active-site inhibitor rather than an allosteric inhibitor (30,39).The crystal structures of malic enzymes reveal that the enzyme is a homotetramer with a dimer of dimers quaternary structure. These structural data establish malic enzymes as a new class of

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confidence: 79%

“…1A) (33). Pigeon c-NADP-ME in complex with NADP ϩ , Mn 2ϩ , and the transition state analog oxalate, as well as Ascaris suum m-NAD-ME in complex with NADH and the inhibitor tartronate, has also been reported (8,9,31). Structural data reveal that the additional exosite does not seem to appear in these two enzymes.…”

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confidence: 99%

Determinants of the Dual Cofactor Specificity and Substrate Cooperativity of the Human Mitochondrial NAD(P)+-dependent Malic Enzyme

Hsieh

Liu

Chang³

et al. 2006

Journal of Biological Chemistry

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“…Various crystal structures of the enzyme in complexes with substrate, metal ion, cofactor, regulator and inhibitor have been successfully resolved since 1999 [6][7][8][9][20][21][22]. There are a total of 14 crystal structures available in the Protein Data Bank.…”

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confidence: 99%

“…In the A. suum m-NAD-MDH, the same position is occupied by a tartronate molecule, which could represent a comparable allosteric binding site [21]. Figure 1(A) shows the fumarate binding site at the dimer interface, which is approx.…”

Section: Introductionmentioning

confidence: 99%

Characterization of the functional role of allosteric site residue Asp102 in the regulatory mechanism of human mitochondrial NAD(P)+-dependent malate dehydrogenase (malic enzyme)

Hung

Kuo

Chang

et al. 2005

Biochemical Journal

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Human mitochondrial NAD(P)+ -dependent malate dehydrogenase (decarboxylating) (malic enzyme) can be specifically and allosterically activated by fumarate. X-ray crystal structures have revealed conformational changes in the enzyme in the absence and in the presence of fumarate. Previous studies have indicated that fumarate is bound to the allosteric pocket via Arg 67 and Arg 91 . Mutation of these residues almost abolishes the activating effect of fumarate. However, these amino acid residues are conserved in some enzymes that are not activated by fumarate, suggesting that there may be additional factors controlling the activation mechanism. In the present study, we tried to delineate the detailed molecular mechanism of activation of the enzyme by fumarate.

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“…1A). In the ME family, Ascaris suum and human m-NAD(P)-ME were found to be activated by fumarate (11,(15)(16)(17)31). However, the relationship between enzyme regulation and subunit-subunit interaction is still unclear.…”

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Functional Roles of the Tetramer Organization of Malic Enzyme

Hsieh¹,

Chen²,

Hung

2009

Journal of Biological Chemistry

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Malic enzyme has a dimer of dimers quaternary structure in which the dimer interface associates more tightly than the tetramer interface. In addition, the enzyme has distinct active sites within each subunit. The mitochondrial NAD(P) ؉ -dependent malic enzyme (m-NAD(P)-ME) isoform behaves cooperatively and allosterically and exhibits a quaternary structure in dimertetramer equilibrium. The cytosolic NADP ؉ -dependent malic enzyme (c-NADP-ME) isoform is noncooperative and nonallosteric and exists as a stable tetramer.In this study, we analyze the essential factors governing the quaternary structure stability for human c-NADP-ME and m-NAD(P)-ME. Site-directed mutagenesis at the dimer and tetramer interfaces was employed to generate a series of dimers of c-NADP-ME and m-NAD(P)-ME. Size distribution analysis demonstrated that human c-NADP-ME exists mainly as a tetramer, whereas human m-NAD(P)-ME exists as a mixture of dimers and tetramers. Kinetic data indicated that the enzyme activity of c-NADP-ME is not affected by disruption of the interface. There are no significant differences in the kinetic properties between AB and AD dimers, and the dimeric form of c-NADP-ME is as active as tetramers. In contrast, disrupting the interface of m-NAD(P)-ME causes the enzyme to be less active than wild type and to become less cooperative for malate binding; the k cat values of mutants decreased with increasing K d,24 values, indicating that the dissociation of subunits at the dimer or tetramer interfaces significantly affects the enzyme activity. The above results suggest that the tetramer is required for a fully functional m-NAD(P)-ME. Taken together, the analytical ultracentrifugation data and the kinetic analysis of these interface mutants demonstrate the differential role of tetramer organization for the c-NADP-ME and m-NAD(P)-ME isoforms. The regulatory mechanism of m-NAD(P)-ME is closely related to the tetramer formation of this isoform.Malic enzymes catalyze a reversible oxidative decarboxylation of L-malate to yield pyruvate and CO 2 with reduction of NAD(P) ϩ to NAD(P)H. This reaction requires a divalent metal ion (Mg 2ϩ or Mn 2ϩ ) for catalysis (1-3). 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). The m-NAD(P)-ME isoform displays dual cofactor specificity; it can use both NAD ϩ and NADP ϩ as the coenzyme, but NAD ϩ is more favored in a physiological environment (6 -8). Dissimilar to the other two isoforms, m-NAD(P)-ME binds malate cooperatively, and it can be allosterically activated by fumarate; the sigmoidal kinetics observed with cooperativity is abo...

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Crystallographic Studies on Ascaris suum NAD-Malic Enzyme Bound to Reduced Cofactor and Identification of an Effector Site

Cited by 32 publications

References 33 publications

Determinants of the Dual Cofactor Specificity and Substrate Cooperativity of the Human Mitochondrial NAD(P)+-dependent Malic Enzyme

Determinants of the Dual Cofactor Specificity and Substrate Cooperativity of the Human Mitochondrial NAD(P)+-dependent Malic Enzyme

Characterization of the functional role of allosteric site residue Asp102 in the regulatory mechanism of human mitochondrial NAD(P)+-dependent malate dehydrogenase (malic enzyme)

Functional Roles of the Tetramer Organization of Malic Enzyme

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