Human NAD(+)-dependent mitochondrial malic enzyme. cDNA cloning, primary structure, and expression in Escherichia coli

Mitochondrial NAD(+)-dependent malic enzyme (EC 1.1.1.40) is expressed in rapidly proliferating cells and tumor cells, where it is probably linked to the conversion of amino acid carbon to pyruvate. In this paper, we report the cDNA cloning, amino acid sequence, and expression in Escherichia coli of functional human NAD(+)-dependent mitochondrial malic enzyme. The cDNA is 1,923 base pairs long and contains an open reading frame coding for a 584-amino acid protein. The molecular mass is 65.4 kDa for the unprocessed precursor protein. Comparison of the amino acid sequence of the human protein with the published NADP(+)-dependent mammalian cytosolic or plant chloroplast malic enzymes reveals highly conserved regions interrupted with long stretches of amino acids without significant homology. Expression of the processed protein in E. coli yielded an enzyme with the same kinetic and allosteric properties as malic enzyme purified from human cells.     

Association of NADP- and NAD-linked malic enzyme acitivities in Zea mays: relation to C4 pathway photosynthesis.

Two of the three metabolic subtypes of species utilizing C₄-pathway photosynthesis are defined by high activities of either NADP malic enzyme (NADP malic enzyme type) or a coenzyme A (CoA)- and acetyl-CoA-activated NAD malic enzyme (NAD malic enzyme type). These enzymes function to decarboxylate malate as an integral part of the photosynthetic process. Leaves of NADP malic enzyme-type species also contain significant NAD-dependent malic enzyme activity. The purpose of the present study was to examine the nature and photosynthetic role of this activity. With Zea mays, this NAD-dependent activity was found to vary widely in fresh leaf extracts. Incubating extracts at 25 °C resulted in a disproportionate increase in NAD activity so that the final ratio of NADP to NAD activity was always about 5. Strong evidence was provided that the NADP and NAD malic enzyme activities in Z. mays extracts were catalyzed by the same enzyme. These activities remained associated during purification and were coincident after polyacrylamide gel electrophoresis. The pH optimum for NAD-dependent activity was about 7.1, compared with 8.3 for NADP malic enzyme activity. Other properties of the NAD-dependent activity are described, a particularly notable feature being the inhibition of this activity by less than 1 μm NADP and NADPH. Evidence is provided that the NADP malic enzyme of several other NADP malic enzyme-type C₄ species also has associated activity toward NAD. We concluded that the NAD-dependent malic enzyme activity would have no significant function in photosynthesis.     

An NAD- and NADP-dependent malic enzyme with regulatory properties in rat liver and adrenal cortex mitochondrial fractions

The NAD- and NADP-dependent malic enzymes from rat liver and adrenal mitochondrial fractions were separated and partially purified by gel filtration on Sepharose 6B. Two activity peaks were observed. The first contained a malic enzyme capable of reducing either NAD or NADP. This enzyme showed sigmoid kinetics in plots of activity versus the malate concentration. Succinate was an allosteric activator and ATP was a competitive inhibitor of malate. The second peak showed hyperbolic kinetics in plots of activity versus the malate concentration and was unaffected by either succinate or ATP. The relative activities of the two malic enzymes were quite constant in the adrenal mitochondrial fractions. In the liver mitochondrial fractions, the activity of the first peak varied and was sometimes absent while the activity of the second peak was quite constant. The kinetic properties of the first malic enzyme implicate it as an important regulator of malate oxidation.     

Characterization of two members of a novel malic enzyme class.

The Gram-negative bacterium Rhizobium meliloti contains two distinct malic enzymes. We report the purification of the two isozymes to homogeneity, and their in vitro characterization. Both enzymes exhibit unusually high subunit molecular weights of about 82 kDa. The NAD(P)(+) specific malic enzyme [EC 1.1.1.39] exhibits positive co-operativity with respect to malate, but Michaelis-Menten type behavior with respect to the co-factors NAD(+) or NADP(+). The enzyme is subject to substrate inhibition, and shows allosteric regulation by acetyl-CoA, an effect that has so far only been described for some NADP(+) dependent malic enzymes. Its activity is positively regulated by succinate and fumarate. In contrast to the NAD(P)(+) specific malic enzyme, the NADP(+) dependent malic enzyme [EC 1.1.1.40] shows Michaelis-Menten type behavior with respect to malate and NADP(+). Apart from product inhibition, the enzyme is not subjected to any regulatory mechanism. Neither reductive carboxylation of pyruvate, nor decarboxylation of oxaloacetate, could be detected for either malic enzyme. Our characterization of the two R. meliloti malic enzymes therefore suggests a number of features uncharacteristic for malic enzymes described so far.     

Activity of pear fruit malic enzyme; its regulation by metabolites

Sigmoid kinetics are reported for the pear malic enzyme (l-malate NADP oxidoreductase, EC 1.1.1.40). Responses have been obtained from possible allosteric effectors. The physiological significance of these responses to metabolites is discussed in relation to a regulatory role of this enzyme in maturation.     

Cytosol malate dehydrogenase in Calicophoron ijimai trematodes and the effect of antiparasitic preparations on its activity

The activity and properties of malate dehydrogenase (MDH; EC 1.1.1.37) and of "malic" enzyme (EC 1.1.1.40) in cytosole of the trematode C. ijimai were determined. The activity of MDH directed to oxaloacetate formation was shown to be 14 times and maximum velocity 13 times lower than that of the reverse reaction. The apparent KM was one order higher in the direct reaction. This confirms the possibility of glycolytic pathway in C. ijimai via CO2 fixation into phosphoenolpyruvate to form oxaloacatate which is readily eliminated by active MDH. The presence of "malic" enzyme in C. ijimai testifies to the occurrence of different pathways of succinate formation in this species.     

Allosteric and isosteric modifiers of NADH binding to cytoplasmic malic dehydrogenase

The binding of NADH to cytoplasmic malic dehydrogenase is shown to be affected by a number of added ligands. One class of ligands appear to be analogs of a substrate for the enzyme, $\text{L}$-malate. These alter the binding constant for NADH without affecting the cooperativity of binding. In contrast, fructose-1,6-diphosphate behaves as an allosteric inhibitor at low enzyme concentrations, apparently by shifting the monomer-dimer equilibrium of the protein to the cooperatively binding dimer. The significance of these results are discussed in terms of a proposed regulatory function for the enzyme.     

A multienzyme complex for CO2 fixation.

Acetyl-coenzyme A carboxylase from Euglena gracilis strain Z was isolated as a component of a multienzyme complex which includes phosphoenolpyruvate carboxylase and malate dehydrogenase. The multienzyme complex was shown to exist in crude extracts and was purified to a homogeneous protein with a molecular weight of 360,000 by gel filtration. The ratio of the activities of the constituent enzymes was acetyl-CoA carboxylase:phosphoenolpyruvate carboxylase:malate dehydrogenase, 1:25:500. The complex is proposed to operate in conjunction with malic enzyme, which is present in Euglena, to facilitate the formation of substrates, malonyl-CoA, and NADPH, for fatty acid biosynthesis. The interaction of the enzymes may represent a means of control of acetyl-CoA carboxylase activity in organisms which do not possess an enzyme subject to allosteric regulation. The acetyl-CoA carboxylase activity from Euglena is unaffected by citrate and isocitrate.     

Isotope effect studies of the chemical mechanism of nicotinamide adenine dinucleotide malic enzyme from Crassula

The 13C primary kinetic isotope effect on the decarboxylation of malate by nicotinamide adenine dinucleotide malic enzyme from Crassula argentea is 1.0199 +/- 0.0006 with proteo L-malate-2-H and 1.0162 +/- 0.0003 with malate-2-d. The primary deuterium isotope effect is 1.45 +/- 0.10 on V/K and 1.93 +/- 0.13 on Vmax. This indicates a stepwise conversion of malate to pyruvate and CO2 with hydride transfer preceding decarboxylation, thereby suggesting a discrete oxaloacetate intermediate. This is in agreement with the stepwise nature of the chemical mechanism of other malic enzymes despite the Crassula enzyme's inability to reduce or decarboxylate oxaloacetate. Differences in morphology and allosteric regulation between enzymes suggest specialization of the Crassula malic enzyme for the physiology of crassulacean acid metabolism while maintaining the catalytic events found in malic enzymes from animal sources.     

Inhibition of the malic enzyme from Acinetobacter calcoaceticus by NADPH and NADH]

The malic enzyme enriched from Acinetobacter calcoaceticus is inhibited by NADPH and NADH. The inhibition afforded by the reduced coenzymes is not affected by NAD+, AMP and 3'.5'-AMP. Against L-malate, NADPH inhibits the enzyme in a noncompetitive linear fashion (Ki = 1.5 x 10(-4) M), against NADP+, competitively linearly (Ki = 5.0 x 10(-5) M). While NADPH acted as a product inhibitor, NADH seems to be an allosteric effector of the malic enzyme, because with L-malate as the variable substrate in the double reciprocal plot, a nonlinear curve is obtained.     

Structural characteristics of the nonallosteric human cytosolic malic enzyme

Human cytosolic NADP⁺-dependent malic enzyme (c-NADP-ME) is neither a cooperative nor an allosteric enzyme, whereas mitochondrial NAD(P)⁺-dependent malic enzyme (m-NAD(P)-ME) is allosterically activated by fumarate. This study examines the molecular basis for the different allosteric properties and quaternary structural stability of m-NAD(P)-ME and c-NADP-ME. Multiple residues corresponding to the fumarate-binding site were mutated in human c-NADP-ME to correspond to those found in human m-NAD(P)-ME. Additionally, the crystal structure of the apo (ligand-free) human c-NADP-ME conformation was determined. Kinetic studies indicated no significant difference between the wild-type and mutant enzymes in K_m,NADP, K_m,malate, and k_cat. A chimeric enzyme, [51-105]_c-NADP-ME, was designed to include the putative fumarate-binding site of m-NAD(P)-ME at the dimer interface of c-NADP-ME; however, this chimera remained nonallosteric. In addition to fumarate activation, the quaternary structural stability of c-NADP-ME and m-NAD(P)-ME is quite different; c-NADP-ME is a stable tetramer, whereas m-NAD(P)-ME exists in equilibrium between a dimer and a tetramer. The quaternary structures for the S57K/N59E/E73K/S102D and S57K/N59E/E73K/S102D/H74K/D78P/D80E/D87G mutants of c-NADP-ME are tetrameric, whereas the K57S/E59N/K73E/D102S m-NAD(P)-ME quadruple mutant is primarily monomeric with some dimer formation. These results strongly suggest that the structural features near the fumarate-binding site and the dimer interface are highly related to the quaternary structural stability of c-NADP-ME and m-NAD(P)-ME. In this study, we attempt to delineate the structural features governing the fumarate-induced allosteric activation of malic enzyme.     

Amino acid substitutions which stabilize aspartate transcarbamoylase in the R state disrupt both homotropic and heterotropic effects

We have used site-specific amino acid substitutions to investigate the linkage between the allosteric properties of arpartate transcarbamoylase and the global conformational transition exhibited by the enzyme upon binding active-site ligands. Two mutationally altered enzymes in which an amino acid substitution had been introduced at a single position in the catalytic polypeptide chain (Lys-164----Glu and Glu-239----Lys) and a third species harboring both of these substitutions (Lys-164:Glu-239----Glu:Lys) were constructed. Sedimentation velocity difference studies were performed in order to assess the effects of the amino acid substitutions on the quaternary structure of the holoenzyme in the absence and presence of various active-site ligands, including the bisubstrate analog, N-(phosphonacetyl)-L-aspartate (PALA), which has been shown previously to promote the allosteric transition. In the absence of ligand, two of the mutationally altered enzymes, Lys-164----Glu and Lys-164:Glu-239----Glu:Lys, existed in the R conformation, isomorphous with that of the PALA-liganded wild-type holoenzyme. These enzymes exhibited no conformational change upon binding PALA. The unliganded Glu-239----Lys enzyme had an average sedimentation coefficient intermediate between that of the unliganded and PALA-liganded states of the wild-type enzyme which could be accounted for in terms of a mixture of T- and R-state molecules. This mutant enzyme was converted to the fully swollen conformation upon binding PALA, phosphate or carbamoyl phosphate. The allosteric properties of the mutationally altered species were investigated by PALA-binding studies and by steady-state enzyme kinetics. In each case, the mutationally altered enzymes were devoid of both homotropic and heterotropic effects, supporting the premise that the allosteric properties of the wild-type enzyme are linked to a ligand-promoted change in quaternary structure.     

Detection of Relationships Between Streptococcus faecalis and Lactobacillus casei by Immunological Studies with Two Forms of Malic Enzyme

Antisera prepared against two isologous malic enzymes from Lactobacillus casei strains 64H and M40 were used to survey and categorize the various malic enzymes found within this diverse species. In addition to detecting three major antigenic variants of malic enzyme within this group, both antisera readily reacted with Streptococcus faecalis malic enzyme. The cross-reactions between the L. casei malic enzyme antisera and the S. faecalis malic enzyme indicated that these iso-functional enzymes found in two apparently diverse groups of organisms were immunologically homologous. A scheme proposing a common ancestry for the two species S. faecalis and L. casei based on the results of quantitative immunological studies is presented.     

Nicotinamide-adenine dinucleotide-linked "malic" enzyme in flight muscle of the tse-tse fly (Glossina) and other insects.

1. A high activity of NAD-linked "malic" enzyme was found in homogenates of flight muscle of different species of tse-tse fly (Glossina). The activity was the same as, or higher than, that of malate dehydrogenase and more than 20-fold that of NADP-linked "malic" enzyme. A similar enzyme was found in the flight muscle of all other insects investigated, but at much lower activities. 2. ACa2+-stimulated oxaloacetate decarboxylase activity was present in all insect flight-muscle preparations investigated, in constant proportion to the NAD-linked "malic" enzyme. 3. A partial purification of the NAD-linked "malic" enzyme from Glossina was effected by DEAE-cellulose chromatography, which separated the enzyme from malate dehydrogenase and NADP-linked "malic" enzyme, but not from oxaloacetate decarboxylase. 4. The intracellular localization of the NAD-linked "malic" enzyme was predominantly mitochondrial; latency studies suggested a localization in the mitochondrial matrix space. 5. Studies on the partially purified enzyme demonstrated that it had a pH optimum between 7.6 and 7.9. It required Mg2+ or Mn2+ for activity; Ca2+ was not effective. The maximum rate was the same with either cation, but the concentration of Mn2+ required was 100 times less than that of Mg2+. Acitivity with NADP was only 1-3% of that with NAD, unless very high (greater than 10mM) concentrations of Mn2+ were present. 6. It is suggested that the NAD-linked "malic" enzyme functions in the proline-oxidation pathway predominant in tse-tse fly flight muscle.     

Purification and characterization of adenosine diphosphate glucose pyrophosphorylase from maize/potato mosaics

Adenosine diphosphate glucose pyrophosphorylase (AGPase) catalyzes a rate-limiting step in starch biosynthesis. The reaction produces ADP-glucose and pyrophosphate from glucose-1-P and ATP. Investigations from a number of laboratories have shown that alterations in allosteric properties as well as heat stability of this enzyme have dramatic positive effects on starch synthesis in the potato (Solanum tuberosum) tuber and seeds of important cereals. Here, we report the characterization of purified recombinant mosaic AGPases derived from protein motifs normally expressed in the maize (Zea mays) endosperm and the potato tuber. These exhibit properties that should be advantageous when expressed in plants. We also present an in-depth characterization of the kinetic and allosteric properties of these purified recombinant AGPases. These data point to previously unrecognized roles for known allosteric effectors.     

Differential characteristics of the cytosol and mitochondrial isozymes of malic enzyme from bovine brain. Effects of dicarboxylic acids and sulfhydryl reagents

The cytosol and mitochondrial isozymes of bovine brain malic enzyme were studied with respect to their sensitivity towards a series of dicarboxylic acids and sulfhydryl reagents. While no effects were obtained with the dicarboxylic acids in the case of the cytosol enzyme, the activity of the mitochondrial variant was increased considerably when either succinate, 2-mercaptosuccinate, or l-aspartate were tested at low concentrations of l-malate. The activation was associated with a clear decrease in the Hill coefficient for l-malate, and this has been taken as an indication of the presence of an allosteric site on the mitochondrial enzyme. The presence of l-malate or a dicarboxylate anion analog is required at this site in order to achieve optimal velocity. The activators were also effective in increasing the reductive carboxylation of pyruvate by the mitochondrial enzyme and had no effect on the cytosol variant. The two isozymes also showed a clear differential sensitivity to 5,5′-dithiobis(2-nitrobenzoic acid) and Hg²⁺, since the mitochondrial malic enzyme was inhibited by concentrations of these reagents far below those required in order to achieve an effect on the activity of the malic enzyme found in the cytosol.     

Structure and function of malic enzymes, a new class of oxidative decarboxylases

Malic enzyme is a tetrameric protein with double dimer structure in which the dimer interface is more intimately contacted than the tetramer interface. Each monomeric unit of the enzyme is composed of four structural domains, which show a different folding topology from those of the other oxidative decarboxylases. The active center is located at the interface between domains B and C. For human mitochondrial malic enzyme, there is an exo nucleotide-binding site for the inhibitor ATP and an allosteric site for the activator fumarate, located at the tetramer and dimer interfaces, respectively. Crystal structures of the enzyme in various complexed forms indicate that the enzyme may exist in equilibrium among two open and two closed forms. Interconversion among these forms involves rigid-body movements of the four structural domains. Substrate binding at the active site shifts the open form to the closed form that represents an active site closure. Fumarate binding at the allosteric site induces the interconversion between forms I and II, which is mediated by the movements of domains A and D. Structures of malic enzyme from different sources are compared with an emphasis on the differences and their implications to structure-function relationships. The binding modes of the substrate, product, cofactors, and transition-state analogue at the active site, as well as ATP and fumarate at the exo site and allosteric site, respectively, provide a clear account for the catalytic mechanism, nucleotide specificities, allosteric regulation, and functional roles of the quaternary structure. The proposed catalytic mechanism involves tyrosine-112 and lysine-183 as the general acid and base, respectively. In addition, a divalent metal ion (Mn(2+) or Mg(2+)) is essential in helping the catalysis. Binding of the metal ion also plays an important role in stabilizing the quaternary structural integrity of the enzyme.     

Purification and Characterization of NAD Malic Enzyme from Leaves of Eleusine coracana and Panicum dichotomiflorum

NAD malic enzyme (EC 1.1.1.39), which is involved in C₄ photosynthesis, was purified to electrophoretic homogeneity from leaves of Eleusine coracana and to near homogeneity from leaves of Panicum dichotomiflorum. The enzyme from each C₄ species was found to have only one type of subunit by SDS polyacrylamide gel electrophoresis. The M_r of subunits of the enzme from E. coracana and P. dichotommiflorum was 63 and 61 kilodaltons, respectively. The native Mr of the enzyme from each species was determined by gel filtration to be about 500 kilodaltons, indicating that the NAD malic enzyme from C₄ species is an octamer of identical subunits. The purified NAD malic enzyme from each C₄ species showed similar kinetic properties with respect to concentrations of malate and NAD; each had a requirement for Mn²⁺ and activation by fructose- 1,6-bisphosphate (FBP) or CoA. A cooperativity with respect to Mn²⁺ was apparent with both enzymes. The activator (FBP) did not change the Hill value but greatly decreased K_0.5 (the concentration giving half-maximal activity) for Mn²⁺. The enzyme from E. coracana showed a very low level of activity when NADP was used as substrate, but this activity was also stimulated by FBP. Significant differences between the enzymes from E. coracana and P. dichotomiflorum were observed in their responses to the activators and their immunochemical properties. The enzyme from E. coracana was largely dependent on the activators FBP or CoA, regardless of concentration of Mn²⁺. In contrast, the enzyme from P. dichotomiflorum showed significant activity in the absence of the activator, especially at high concentrations of Mn²⁺. Both immunodiffusion and immunoprecipitation, using antiserum raised against the purified NAD malic enzyme from E. coracana, revealed partial antigenic differences between the enzymes from E. coracana and P. dichotomiflorum. The activity of the NAD malic enzyme from Amaranthus edulis, a typical NAD malic enzyme type C₄ dicot, was not inhibited by the antiserum raised against the NAD malic enzyme from E. coracana.     

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 CO2 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 A resolution by the selenomethionyl multiwavelength anomalous diffraction method and refined to 2.1 A 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 A away from the active site. The natural ligand for this second binding site may be ATP, an allosteric inhibitor of the enzyme.