Identification of an NAD(P)+-dependent 'malic' enzyme in small-intestinal-mucosal mitochondria.
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ABSTRACT:
SUBMITTER: Sauer LA
PROVIDER: S-EPMC1161692 | biostudies-other | 1979 Oct
REPOSITORIES: biostudies-other
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Project description:Here, we found a natural compound, embonic acid (EA), that can specifically inhibit the enzymatic activity of mitochondrial NAD(P)+-dependent malic enzyme (m-NAD(P)-ME, ME2) either in vitro or in vivo. The in vitro IC50 value of EA for m-NAD(P)-ME was 1.4 ± 0.4 μM. Mutagenesis and binding studies revealed that the putative binding site of EA on m-NAD(P)-ME is located at the fumarate binding site or at the dimer interface near the site. Inhibition studies reveal that EA displayed a non-competitive inhibition pattern, which demonstrated that the binding site of EA was distinct from the active site of the enzyme. Therefore, EA is thought to be an allosteric inhibitor of m-NAD(P)-ME. Both EA treatment and knockdown of m-NAD(P)-ME by shRNA inhibited the growth of H1299 cancer cells. The protein expression and mRNA synthesis of m-NAD(P)-ME in H1299 cells were not influenced by EA, suggesting that the EA-inhibited H1299 cell growth occurs through the suppression of in vivo m-NAD(P)-ME activity EA treatment further induced the cellular senescence of H1299 cells. However, down-regulation of the enzyme-induced cellular senescence was not through p53. Therefore, the EA-evoked senescence of H1299 cells may occur directly through the inhibition of ME2 or a p53-independent pathway.
| S-EPMC4652989 | biostudies-literature
Project description:BACKGROUND:Anopheles stephensi mitochondrial malic enzyme (ME) emerged as having a relevant role in the provision of pyruvate for the Krebs' cycle because inhibition of this enzyme results in the complete abrogation of oxygen uptake by mitochondria. Therefore, the identification of ME in mitochondria from immortalized A. stephensi (ASE) cells and the investigation of the stereoselectivity of malate analogues are relevant in understanding the physiological role of ME in cells of this important malaria parasite vector and its potential as a possible novel target for insecticide development. METHODS:To characterize the mitochondrial ME from immortalized ASE cells (Mos. 43; ASE), mass spectrometry analyses of trypsin fragments of ME, genomic sequence analysis and biochemical assays were performed to identify the enzyme and evaluate its activity in terms of cofactor dependency and inhibitor preference. RESULTS:The encoding gene sequence and primary sequences of several peptides from mitochondrial ME were found to be highly homologous to the mitochondrial ME from Anopheles gambiae (98%) and 59% homologous to the mitochondrial NADP+-dependent ME isoform from Homo sapiens. Measurements of ME activity in mosquito mitochondria isolated from ASE cells showed that (i) Vmax with NAD+ was 3-fold higher than that with NADP+, (ii) addition of Mg2+ or Mn2+ increased the Vmax by 9- to 21-fold, with Mn2+ 2.3-fold more effective than Mg2+, (iii) succinate and fumarate increased the activity by 2- and 5-fold, respectively, at sub-saturating concentrations of malate, (iv) among the analogs of L-malate tested as inhibitors of the NAD+-dependent ME catalyzed reaction, small (2- to 3-carbons) organic diacids carrying a 2-hydroxyl/keto group behaved as the most potent inhibitors of ME activity (e.g., oxaloacetate, tartronic acid and oxalate). CONCLUSIONS:The biochemical characterization of Anopheles stephensi ME is of critical relevance given its important role in bioenergetics, suggesting that it is a suitable target for insecticide development.
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Project description:Malic enzyme (ME) comprises a family of proteins with multiple isoforms located in different compartments of eukaryotic cells. In plants, cytosolic and plastidic enzymes share several characteristics such as NADP specificity (NADP-ME), oxaloacetate decarboxylase (OAD) activity, and homo-oligomeric assembly. However, mitochondrial counterparts are NAD-dependent proteins (mNAD-ME) lacking OAD activity, which can be structured as homo- and hetero-oligomers of two different subunits. In this study, we examined the molecular basis of these differences using multiple sequence analysis, structural modeling, and phylogenetic approaches. Plant mNAD-MEs show the lowest identity values when compared with other eukaryotic MEs with major differences including short amino acid insertions distributed throughout the primary sequence. Some residues in these exclusive segments are co-evolutionarily connected, suggesting that they could be important for enzymatic functionality. Phylogenetic analysis indicates that eukaryotes from different kingdoms used different strategies for acquiring the current set of NAD(P)-ME isoforms. In this sense, while the full gene family of vertebrates derives from the same ancestral gene, plant NADP-ME and NAD-ME isoforms have a distinct evolutionary history. Plant NADP-ME genes may have arisen from the α-protobacterial-like mitochondrial ancestor, a characteristic shared with major eukaryotic taxa. On the other hand, plant mNAD-ME genes were probably gained through an independent process involving the Archaeplastida ancestor. Finally, several residue signatures unique to all plant mNAD-MEs could be identified, some of which might be functionally connected to their exclusive biochemical properties. In light of these results, molecular evolutionary scenarios for these widely distributed enzymes in plants are discussed.
| S-EPMC5958461 | biostudies-literature
Project description:The metabolic program of osteoblasts, the chief bone-making cells, remains incompletely understood. Here in murine calvarial cells, we establish that osteoblast differentiation under aerobic conditions is coupled with a marked increase in glucose consumption and lactate production but reduced oxygen consumption. As a result, aerobic glycolysis accounts for approximately 80% of the ATP production in mature osteoblasts. In vivo tracing with 13C-labeled glucose in the mouse shows that glucose in bone is readily metabolized to lactate but not organic acids in the TCA cycle. Glucose tracing in osteoblast cultures reveals that pyruvate is carboxylated to form malate integral to the malate-aspartate shuttle. RNA sequencing (RNA-seq) identifies Me2, encoding the mitochondrial NAD-dependent isoform of malic enzyme, as being specifically upregulated during osteoblast differentiation. Knockdown of Me2 markedly reduces the glycolytic flux and impairs osteoblast proliferation and differentiation. Thus, the mitochondrial malic enzyme functionally couples the mitochondria with aerobic glycolysis in osteoblasts.
| S-EPMC8183612 | biostudies-literature
Project description:Human mitochondrial NAD(P)+-dependent malic enzyme (ME2) is well recognized to associate with cancer cell metabolism, and the single nucleotide variants (SNVs) of ME2 may play a role in enzyme regulation. Here we reported that the SNVs of ME2 occurring in the allosteric sites lead to inactivation or overactivation of ME2. Two ME2-SNVs, ME2_R67Q and ME2-R484W, that demonstrated inactivating or overactivating enzyme activities of ME2, respectively, have different impact toward the cells. The cells with overactivating SNV enzyme, ME2_R484W, grow more rapidly and are more resistant to cellular senescence than the cells with wild-type or inactivating SNV enzyme, ME2_R67Q. Crystal structures of these two ME2-SNVs reveal that ME2_R67Q was an inactivating "dead form," and ME2_R484W was an overactivating "closed form" of the enzyme. The resolved ME2-SNV structures provide a molecular basis to explain the abnormal kinetic properties of these SNV enzymes.
| S-EPMC7847962 | biostudies-literature
Project description:Human mitochondrial NAD(P)+-dependent malic enzyme (m-NAD(P)-ME) has a dimer of dimers quaternary structure with two independent allosteric sites in each monomer. Here, we reveal the different effects of nucleotide ligands on the quaternary structure regulation and functional role of the human m-NAD(P)-ME exosite. In this study, size distribution analysis was utilized to investigate the monomer-dimer-tetramer equilibrium of m-NAD(P)-ME in the presence of different ligands, and the monomer-dimer (Kd,12) and dimer-tetramer (Kd,24) dissociation constants were determined with these ligands. With NAD+, the enzyme formed more tetramers, and its Kd,24 (0.06 µM) was 6-fold lower than the apoenzyme Kd,24 (0.34 µM). When ATP was present, the enzyme displayed more dimers, and its Kd,24 (2.74 µM) was 8-fold higher than the apoenzyme. Similar to the apoenzyme, the ADP-bound enzyme was present as a tetramer with a small amount of dimers and monomers. These results indicate that NAD+ promotes association of the dimeric enzyme into tetramers, whereas ATP stimulates dissociation of the tetrameric enzyme into dimers, and ADP has little effect on the tetrameric stability of the enzyme. A series of exosite mutants were created using site-directed mutagenesis. Size distribution analysis and kinetic studies of these mutants with NAD+ or ATP indicated that Arg197, Asn482 and Arg556 are essential for the ATP binding and ATP-induced dissociation of human m-NAD(P)-ME. In summary, the present results demonstrate that nucleotides perform discrete functions regulating the quaternary structure and catalysis of m-NAD(P)-ME. Such regulation by the binding of different nucleotides may be critically associated with the physiological concentrations of these ligands.
| S-EPMC6591397 | biostudies-literature
Project description:Malic enzymes are oxidative decarboxylases with NAD(+) or NAD(P)(+) as cofactor that catalyze the conversion of L-malate to pyruvate and CO2. The aim of this study was to discover and characterize a potent inhibitor of human NAD(P)(+)-dependent malic enzyme 2 (ME2).Recombinant human ME2-His-Tag fusion protein was overexpressed in E coli and purified with Ni-NTA resin. A high-throughput screening (HTS) assay was developed to find ME2 inhibitors. Detergent Brij-35 was used to exclude false positives. The characteristics of the inhibitor were analyzed with enzyme kinetics analysis. A thermal shift assay for ME2 was carried out to verify the binding of the inhibitor with the enzyme.An HTS system for discovering ME2 inhibitors was established with a Z' factor value of 0.775 and a signal-to-noise ratio (S/N) of 9.80. A library containing 12 683 natural products was screened. From 47 hits, NPD387 was identified as an inhibitor of ME2. The primary structure-activity relationship study on NPD387 derivatives showed that one derivative NPD389 was more potent than the parent compound NPD387 (the IC50 of NPD389 was 4.63 ± 0.36 μmol/L or 5.59 ± 0.38 μmol/L, respectively, in the absence or presence of 0.01% Brij-35 in the assay system). The enzyme kinetics analysis showed that NPD389 was a fast-binding uncompetitive inhibitor with respect to the substrate NAD(+) and a mixed-type inhibitor with respect to the substrate L-malate.NPD389 is a potent ME2 inhibitor that binds to the enzyme in a fast-binding mode, acting as an uncompetitive inhibitor with respect to the substrate NAD(+) and a mixed-type inhibitor with respect to the substrate L-malate.
| S-EPMC4814031 | biostudies-other
Project description:NADP+-dependent malic enzyme 1 (ME1) decarboxylates malate to form pyruvate and NADPH in the cytoplasm, where it mediates diverse biological functions related to the generation of lipids and other cellular building blocks. As such, ME1 has been implicated in the progression of cancers and has received attention as a promising drug target. Here we report the identification of a novel small-molecule inhibitor of ME1, designated AS1134900. AS1134900 is highly selective for ME1 compared with ME2 and uncompetitively inhibits ME1 activity in the presence of its substrates NADP+ and malate. In addition, X-ray crystal structure analysis of the enzyme-inhibitor complex revealed that AS1134900 binds outside the ME1 active site in a novel allosteric site. Structural comparison of the ME1 quaternary complex with AS1134900, NADPH, and Mn2+, alongside known crystal structures of malic enzymes, indicated the determined crystal ME1-inhibitor complex is in the open form conformation. These results provide insights and a starting point for further discovery of drugs that inhibit ME1 activity in cancer cells.
| S-EPMC9352307 | biostudies-literature
Project description: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 Arg67 and Arg91. 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. Site-directed mutagenesis was used to replace Asp102, which is one of the charged amino acids in the fumarate binding pocket and is not conserved in other decarboxylating malate dehydrogenases. In order to explore the charge effect of this residue, Asp102 was replaced by alanine, glutamate or lysine. Our experimental data clearly indicate the importance of Asp102 for activation by fumarate. Mutation of Asp102 to Ala or Lys significantly attenuated the activating effect of fumarate on the enzyme. Kinetic parameters indicate that the effect of fumarate was mainly to decrease the K(m) values for malate, Mg2+ and NAD+, but it did not notably elevate kcat. The apparent substrate K(m) values were reduced by increasing concentrations of fumarate. Furthermore, the greatest effect of fumarate activation was apparent at low malate, Mg2+ or NAD+ concentrations. The K(act) values were reduced with increasing concentrations of malate, Mg2+ and NAD+. The Asp102 mutants, however, are much less sensitive to regulation by fumarate. Mutation of Asp102 leads to the desensitization of the co-operative effect between fumarate and substrates of the enzyme.
| S-EPMC1317662 | biostudies-literature
Project description:In Cleomaceae species, NAD-malic enzyme (NAD-ME) was independently co-opted to participate in C4 photosynthesis. In C4 Cleome species, all NAD-ME genes (NAD-MEα, -ß1 and -ß2) were affected by C4 evolution and are expressed at higher levels than their C3 orthologs in Tarenaya hassleriana. In C3 Cleome, the NAD-ME housekeeping function is performed by two heteromers, NAD-MEα/ß1 and NAD-MEα/ß2, with similar biochemical properties and tissue occurrence. In the C4 species, Gynandropsis gynandra and Cleome angustifolia, this role is performed only by the NAD-MEα/ß2 heteromer. In these C4 species NAD-MEα/ß1 is preferentially present in leaves of the C4 species where is the predominant isoform. GgNAD-MEα/ß1 exhibits high catalytic efficiency and is differentially activated by the C4 intermediate aspartate. In C4 Cleome NAD-MEα/ß1 represents thus the C4-decarboxylase. GgNAD-MEß1and CaNAD-MEß1 are non-catalytic subunits but impart a stabilizing effect on the associated α-subunit. We conclude that in C4 Cleome the functions of NAD-ME as a TCA cycle associated enzyme and as a C4 photosynthetic decarboxylase coexist in BSC mitochondria and are performed by isoforms originated through associations of differentially adapted subunits.
2021-11-30 | PXD026547 | Pride