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Huichih Hung - One of the best experts on this subject based on the ideXlab platform.

  • human mitochondrial nad p dependent Malic Enzyme participates in cutaneous melanoma progression and invasion
    Journal of Investigative Dermatology, 2015
    Co-Authors: Guangyaw Liu, Yunglung Chang, Hongwei Gao, Chienping Chiang, Weiming Wang, Shihming Huang, Huichih Hung
    Abstract:

    Cutaneous melanoma is the most life-threatening neoplasm of the skin, accounting for most of the skin cancer deaths. Accumulating evidence suggests that targeting metabolism is an appealing strategy for melanoma therapy. Mitochondrial NAD(P) + –dependent Malic Enzyme (ME2), an oxidative decarboxylase, was evaluated for its biological significance in cutaneous melanoma progression. ME2 mRNA and protein expression significantly increased during melanoma progression, as evidenced by Gene Expression Omnibus analysis and immunohistochemistry on clinically annotated tissue microarrays, respectively. In addition, ME2 knockdown attenuated melanoma cell proliferation in vitro . ME2 ablation resulted in reduced cellular ATP levels and elevated cellular reactive oxygen species production, which activated the AMP-activated protein kinase pathway and inhibited acetyl-CoA carboxylase. Furthermore, ME2 expression was associated with cell migration and invasion. ME2 knockdown decreased anchorage-independent growth in vitro and tumor cell growth in vivo. These results suggested that ME2 might be an important factor in melanoma progression and a novel biomarker of invasion.

  • structural characteristics of the nonallosteric human cytosolic Malic Enzyme
    Biochimica et Biophysica Acta, 2014
    Co-Authors: Juyi Hsieh, Mengchun Chen, Paichun Yang, Huiyi Chen, Neili Chan, Jyunghurng Liu, Huichih Hung
    Abstract:

    Abstract 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 Km,NADP, Km,malate, and kcat. 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.

  • influential factor contributing to the isoform specific inhibition by atp of human mitochondrial nad p dependent Malic Enzyme functional roles of the nucleotide binding site lys346
    FEBS Journal, 2008
    Co-Authors: Juyi Hsieh, Guangyaw Liu, Huichih Hung
    Abstract:

    Human mitochondrial NAD(P)+-dependent Malic Enzyme (m-NAD-ME) is a Malic Enzyme isoform with dual cofactor specificity, ATP inhibition and substrate cooperativity. The determinant of ATP inhibition in Malic Enzyme isoforms has not yet been identified. Sequence alignment of nucleotide-binding sites of ME isoforms revealed that Lys346 is conserved uniquely in m-NAD-ME. In other ME isoforms, this residue is serine. As the inhibitory effect of ATP is more pronounced on m-NAD-ME than on other ME isoforms, we have examined the possible role of Lys346 by replacing it to alanine, serine or arginine. Our kinetic data indicate that the K346S mutant Enzyme displays a shift in its cofactor preference from NAD+ to NADP+ upon increasing kcat,NADP and decreasing Km,NADP. Furthermore, the cooperative binding of malate becomes less significant in human m-NAD-ME after mutation of Lys346. The h value for the wild-type is close to 2, but those of the K346 mutants are approximately 1.5. The K346 mutants can also be activated by fumarate and the cooperative effect can be abolished by fumarate, suggesting that the allosteric property is retained in these mutants. Our data strongly suggest that Lys346 in human m-NAD-ME is required for ATP inhibition. Mutation of Lys346 to Ser or Ala causes the Enzyme to be much less sensitive to ATP, similar to cytosolic NADP-dependent Malic Enzyme. Substitution of Lys to Arg did not change the isoform-specific inhibition of the Enzyme by ATP. The inhibition constants of ATP are increased for K346S and K346A, but are similar to those of the wild-type for K346R, suggesting that the positive charge rather than group specificity is required for binding affinity of ATP. Thus, ATP inhibition is proposed to be determined by the electrostatic potential involving the positive charge on the side chain of Lys346.

  • determinants of the dual cofactor specificity and substrate cooperativity of the human mitochondrial nad p dependent Malic Enzyme functional roles of glutamine 362
    Journal of Biological Chemistry, 2006
    Co-Authors: Juyi Hsieh, Gu-gang Chang, Guangyaw Liu, Huichih Hung
    Abstract:

    The human mitochondrial NAD(P)+-dependent Malic Enzyme (m-NAD-ME) is a Malic Enzyme isoform with dual cofactor specificity and substrate binding cooperativity. Previous kinetic studies have suggested that Lys362 in the pigeon cytosolic NADP+-dependent Malic Enzyme has remarkable effects on the binding of NADP+ to the Enzyme and on the catalytic power of the Enzyme (Kuo, C. C., Tsai, L. C., Chin, T. Y., Chang, G.-G., and Chou, W. Y. (2000) Biochem. Biophys. Res. Commun. 270, 821-825). In this study, we investigate the important role of Gln362 in the transformation of cofactor specificity from NAD+ to NADP+ in human m-NAD-ME. Our kinetic data clearly indicate that the Q362K mutant shifted its cofactor preference from NAD+ to NADP+. The Km(NADP) and kcat(NADP) values for this mutant were reduced by 4-6-fold and increased by 5-10-fold, respectively, compared with those for the wild-type Enzyme. Furthermore, up to a 2-fold reduction in Km(NADP)/Km(NAD) and elevation of kcat(NADP)/kcat(NAD) were observed for the Q362K Enzyme. Mutation of Gln362 to Ala or Asn did not shift its cofactor preference. The Km(NADP)/Km(NAD) and kcat(NADP)/kcat(NAD) values for Q362A and Q362N were comparable with those for the wild-type Enzyme. The ΔG values for Q362A and Q362N with either NAD+ or NADP+ were positive, indicating that substitution of Gln with Ala or Asn at position 362 brings about unfavorable cofactor binding at the active site and thus significantly reduces the catalytic efficiency. Our data also indicate that the cooperative binding of malate became insignificant in human m-NAD-ME upon mutation of Gln362 to Lys because the sigmoidal phenomenon appearing in the wild-type Enzyme was much less obvious that that in Q362K. Therefore, mutation of Gln362 to Lys in human m-NAD-ME alters its kinetic properties of cofactor preference, malate binding cooperativity, and allosteric regulation by fumarate. However, the other Gln362 mutants, Q362A and Q362N, have conserved malate binding cooperativity and NAD+ specificity. In this study, we provide clear evidence that the single mutation of Gln362 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.

Carlos S Andreo - One of the best experts on this subject based on the ideXlab platform.

  • identification of domains involved in tetramerization and malate inhibition of maize c4 nadp Malic Enzyme
    Journal of Biological Chemistry, 2007
    Co-Authors: Enrique Detarsio, Mariana Saigo, Carlos S Andreo, Clarisa Ester Alvarez, Maria F Drincovich
    Abstract:

    C4 photosynthetic NADP-Malic Enzyme (ME) has evolved from non-C4 isoforms and gained unique kinetic and structural properties during this process. To identify the domains responsible for the structural and kinetic differences between maize C4 and non-C4-NADP-ME several chimeras between these isoforms were constructed and analyzed. By using this approach, we found that the region flanked by amino acid residues 102 and 247 is critical for the tetrameric state of C4-NADP-ME. In this way, the oligomerization strategy of these NADP-ME isoforms differs markedly from the one that present non-plant NADP-ME with known crystal structures. On the other hand, the region from residue 248 to the C-terminal end of the C4 isoform is involved in the inhibition by high malate concentrations at pH 7.0. The inhibition pattern of the C4-NADP-ME and some of the chimeras suggested an allosteric site responsible for such behavior. This pH-dependent inhibition could be important for regulation of the C4 isoform in vivo, with the Enzyme presenting maximum activity while photosynthesis is in progress.

  • maize recombinant non c4 nadp Malic Enzyme a novel dimeric Malic Enzyme with high specific activity
    Plant Molecular Biology, 2004
    Co-Authors: Mariana Saigo, Veronica G Maurino, Carlos S Andreo, Federico P Bologna, Enrique Detarsio, Maria F Drincovich
    Abstract:

    Among the different isoforms of NADP-Malic Enzyme (NADP-ME) involved in a wide range of metabolic pathways in plants, the NADP-ME that participates in C4-photosynthesis is the most studied. In the present work, the expression in E. coli of a cDNA encoding for a maize non-photosynthetic NADP-ME is presented. The recombinant NADP-ME thus obtained presents kinetic and structural properties different from the Enzyme previously purified from etiolated leaves and roots. Moreover, the recombinant non-photosynthetic NADP-ME presents very high intrinsic NADP-ME activity, which is unexpected for a non-C4 NADP-ME. Using antibodies against this recombinant Enzyme, an immunoreactive band of 66 kDa is detected in different maize tissues indicating that the 66 kDa-NADP-ME is in fact a protein expressed in␣vivo. The recombinant NADP-ME assembles as a dimer, although the results obtained indicate that a higher molecular mass oligomeric state of the Enzyme is found in maize roots in vivo. In this way, maize presents at least three NADP-ME isoforms: a 72 kDa constitutive form (previously characterized); the novel non-photosynthetic 66 kDa isoform characterized in this work (which is the product of the ZmChlMe2gene and the likely precursor to the evolution of the photosynthetic C4 NADP-ME) and the 62 kDa isoform (implicated in C4 photosynthesis). The contribution of the present work anticipates further studies concerning the equilibrium between the oligomeric states of the NADP-ME isoforms and the evolution towards the C4 isoEnzyme in maize.

  • nadp Malic Enzyme from plants a ubiquitous Enzyme involved in different metabolic pathways
    FEBS Letters, 2001
    Co-Authors: Maria F Drincovich, Paula Casati, Carlos S Andreo
    Abstract:

    NADP-Malic Enzyme (NADP-ME) is a widely distributed Enzyme that catalyzes the oxidative decarboxylation of L-malate. Photosynthetic NADP-MEs are found in C4 bundle sheath chloroplasts and in the cytosol of CAM plants, while non-photosynthetic NADP-MEs are either plastidic or cytosolic in various plants. We propose a classification of plant NADP-MEs based on their physiological function and localization and we describe recent advances in the characterization of each isoform. Based on the alignment of amino acid sequences of plant NADP-MEs, we identify putative binding sites for the substrates and analyze the phylogenetic origin of each isoform, revealing several features of the molecular evolution of this ubiquitous Enzyme.

  • Malate metabolism by NADP-Malic Enzyme in plant defense
    Photosynthesis Research, 1999
    Co-Authors: Paula Casati, Maria F Drincovich, Gerald E Edwards, Carlos S Andreo
    Abstract:

    Malate is involved in various metabolic pathways, and there are several Enzymes that metabolize it. One important malate metabolizing Enzyme is NADP-Malic Enzyme (NADP-ME). NADP-ME functions in many different pathways in plants, having an important role in C_4 photosynthesis where it releases the CO_2 to be used in carbon fixation by Rubisco. Apart from this specialized role, NADP-ME is thought to fulfill diverse housekeeping functions because of its universal presence in different plant tissues. NADP-ME is induced after wounding or exposure to UV-B radiation. In this way, the Enzyme is implicated in defense-related deposition of lignin by providing NADPH for the two NADPH-dependent reductive steps in monolignol biosynthesis. On the other hand, it can supply NADPH for flavonoid biosynthesis as many steps in the flavonoid biosynthesis pathway require reductive power. Pyruvate, another product of NADP-ME reaction, can be used for obtaining ATP through respiration in the mitochondria; and may serve as a precursor for synthesis of phosphoenolpyruvate (PEP). PEP is utilized in the shikimate pathway, leading to the synthesis of aromatic amino acids including phenylalanine, the common substrate for lignin and flavonoid synthesis. Moreover, NADP-ME can be involved in mechanisms producing NADPH for synthesis of activated oxygen species that are produced in order to kill or damage pathogens. In conclusion, an increase in the levels of NADP-ME could provide building blocks and energy for biosynthesis of defense compounds, suggesting a role of malate metabolism in plant defense.

  • evolution of c4 photosynthesis in flaveria species isoforms of nadp Malic Enzyme
    Plant Physiology, 1998
    Co-Authors: Maria F Drincovich, Paula Casati, Carlos S Andreo, Vincent R Franceschi, Saul J Chessin, Gerald E Edwards
    Abstract:

    NADP-Malic Enzyme (NADP-ME, EC 1.1.1.40), a key Enzyme in C4 photosynthesis, provides CO2 to the bundle-sheath chloroplasts, where it is fixed by ribulose-1,5-bisphosphate carboxylase/oxygenase. We characterized the isoform pattern of NADP-ME in different photosynthetic species of Flaveria (C3, C3-C4 intermediate, C4-like, C4) based on sucrose density gradient centrifugation and isoelectric focusing of the native protein, western-blot analysis of the denatured protein, and in situ immunolocalization with antibody against the 62-kD C4 isoform of maize. A 72-kD isoform, present to varying degrees in all species examined, is predominant in leaves of C3 Flaveria spp. and is also present in stem and root tissue. By immunolabeling, NADP-ME was found to be mostly localized in the upper palisade mesophyll chloroplasts of C3 photosynthetic tissue. Two other isoforms of the Enzyme, with molecular masses of 62 and 64 kD, occur in leaves of certain intermediates having C4 cycle activity. The 62-kD isoform, which is the predominant highly active form in the C4 species, is localized in bundle-sheath chloroplasts. Among Flaveria spp. there is a 72-kD constitutive form, a 64-kD form that may have appeared during evolution of C4 metabolism, and a 62-kD form that is necessary for the complete functioning of C4 photosynthesis.

Wei Chen - One of the best experts on this subject based on the ideXlab platform.

  • increased fatty acid unsaturation and production of arachidonic acid by homologous over expression of the mitochondrial Malic Enzyme in mortierella alpina
    Biotechnology Letters, 2014
    Co-Authors: Guangfei Hao, Haiqin Chen, Hao Zhang, Wei Chen, Xiaoyun Huang
    Abstract:

    Malic Enzyme (ME) catalyses the oxidative decarboxylation of l-malate to pyruvate and provides NADPH for intracellular metabolism, such as fatty acid synthesis. Here, the mitochondrial ME (mME) gene from Mortierella alpina was homologously over-expressed. Compared with controls, fungal arachidonic acid (ARA; 20:4 n−6) content increased by 60 % without affecting the total fatty acid content. Our results suggest that enhancing mME activity may be an effective mean to increase industrial production of ARA in M. alpina.

  • role of Malic Enzyme during fatty acid synthesis in the oleaginous fungus mortierella alpina
    Applied and Environmental Microbiology, 2014
    Co-Authors: Zhennan Gu, Haiqin Chen, Hao Zhang, Wei Chen
    Abstract:

    The generation of NADPH by Malic Enzyme (ME) was postulated to be a rate-limiting step during fatty acid synthesis in oleaginous fungi, based primarily on the results from research focusing on ME in Mucor circinelloides. This hypothesis is challenged by a recent study showing that leucine metabolism, rather than ME, is critical for fatty acid synthesis in M. circinelloides. To clarify this, the gene encoding ME isoform E from Mortierella alpina was homologously expressed. ME overexpression increased the fatty acid content by 30% compared to that for a control. Our results suggest that ME may not be the sole rate-limiting Enzyme, but does play a role, during fatty acid synthesis in oleaginous fungi.

  • regulatory properties of Malic Enzyme in the oleaginous yeast yarrowia lipolytica and its non involvement in lipid accumulation
    Biotechnology Letters, 2013
    Co-Authors: H Zhang, Haiqin Chen, Yuanda Song, Colin Ratledge, Luning Zhang, Yong Q Chen, Wei Chen
    Abstract:

    Malic Enzyme (EC 1.1.1.40) converts l-malate to pyruvate and CO2 providing NADPH for metabolism especially for lipid biosynthesis in oleaginous microorganisms. However, its role in the oleaginous yeast, Yarrowia lipolytica, is unclear. We have cloned the Malic Enzyme gene (YALI0E18634g) from Y. lipolytica into pET28a, expressed it in Escherichia coli and purified the recombinant protein (YlME). YlME used NAD+ as the primary cofactor. Km values for NAD+ and NADP+ were 0.63 and 3.9 mM, respectively. Citrate, isocitrate and α-ketoglutaric acid (>5 mM) were inhibitory while succinate (5–15 mM) increased NADP+- but not NAD+-dependent activity. To determine if fatty acid biosynthesis could be increased in Y. lipolytica by providing additional NADPH from an NADP+-dependent Malic Enzyme, the Malic Enzyme gene (mce2) from an oleaginous fungus, Mortierella alpina, was expressed in Y. lipolytica. No significant changes occurred in lipid content or fatty acid profiles suggesting that Malic Enzyme is not the main source of NADPH for lipid accumulation in Y. lipolytica.

  • annotation and analysis of Malic Enzyme genes encoding for multiple isoforms in the fungus mucor circinelloides cbs 277 49
    Biotechnology Letters, 2012
    Co-Authors: Wanwipa Vongsangnak, Wei Chen, Colin Ratledge, Yingtong Zhang, Yuanda Song
    Abstract:

    Based on the newly-released genomic data of Mucor circinelloides CBS 277.49, we have annotated five genes encoding for Malic Enzyme: all code for proteins that contain conserved domains/motifs for Malic acid binding, NAD+ binding and NAD(P)+ binding. Phylogenetic analysis for Malic Enzyme genes showed that genes ID 78524 and 11639 share ~80% amino acid identity and are grouped in cluster 1; genes ID 182779, 186772 and 116127 share ~66% amino acid identity are grouped in cluster 2. Genes ID 78524, 11639 and 166127 produce proteins that are localized in the mitochondrion, while the products from genes 182779 and 186772 are localized in the cytosol. Based on the comparative analysis published previously by Song et al. (Microbiology 147:1507–1515, 2001), we propose that Malic Enzyme genes ID 78524, 166127, 182779, 186772, 11639, respectively, represent protein isoforms I, II, III/IV, V, and VI.

Juyi Hsieh - One of the best experts on this subject based on the ideXlab platform.

  • single nucleotide variants lead to dysregulation of the human mitochondrial nad p dependent Malic Enzyme
    iScience, 2021
    Co-Authors: Juyi Hsieh, Haoping Yang, Sunil Kumar Tewary, Huichen Cheng, Yiliang Liu, Shihchieh Tai, Weilin Chen, Chienhui Hsu
    Abstract:

    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.

  • Fumarate Analogs Act as Allosteric Inhibitors of the Human Mitochondrial NAD(P)+-Dependent Malic Enzyme
    2015
    Co-Authors: Juyi Hsieh, Liu Jyung-hurng, Yang Pai-chun, Lin Chi-li, Liu Guang-yaw, Hung Hui-chih
    Abstract:

    Human mitochondrial NAD(P)+-dependent Malic Enzyme (m-NAD(P)-ME) is allosterically activated by the four-carbon trans dicarboxylic acid, fumarate. Previous studies have suggested that the dicarboxylic acid in a trans conformation around the carbon-carbon double bond is required for the allosteric activation of the Enzyme. In this paper, the allosteric effects of fumarate analogs on m-NAD(P)-ME are investigated. Two fumarate-insensitive mutants, m-NAD(P)-ME_R67A/R91A and m-NAD(P)-ME_K57S/E59N/K73E/D102S, as well as c-NADP-ME, were used as the negative controls. Among these analogs, mesaconate, trans-aconitate, monomethyl fumarate and monoethyl fumarate were allosteric activators of the Enzyme, while oxaloacetate, diethyl oxalacetate, and dimethyl fumarate were found to be allosteric inhibitors of human m-NAD(P)-ME. The IC50 value for diethyl oxalacetate was approximately 2.5 mM. This paper suggests that the allosteric inhibitors may impede the conformational change from open form to closed form and therefore inhibit m-NAD(P)-ME Enzyme activity

  • structural characteristics of the nonallosteric human cytosolic Malic Enzyme
    Biochimica et Biophysica Acta, 2014
    Co-Authors: Juyi Hsieh, Mengchun Chen, Paichun Yang, Huiyi Chen, Neili Chan, Jyunghurng Liu, Huichih Hung
    Abstract:

    Abstract 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 Km,NADP, Km,malate, and kcat. 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.

  • influential factor contributing to the isoform specific inhibition by atp of human mitochondrial nad p dependent Malic Enzyme functional roles of the nucleotide binding site lys346
    FEBS Journal, 2008
    Co-Authors: Juyi Hsieh, Guangyaw Liu, Huichih Hung
    Abstract:

    Human mitochondrial NAD(P)+-dependent Malic Enzyme (m-NAD-ME) is a Malic Enzyme isoform with dual cofactor specificity, ATP inhibition and substrate cooperativity. The determinant of ATP inhibition in Malic Enzyme isoforms has not yet been identified. Sequence alignment of nucleotide-binding sites of ME isoforms revealed that Lys346 is conserved uniquely in m-NAD-ME. In other ME isoforms, this residue is serine. As the inhibitory effect of ATP is more pronounced on m-NAD-ME than on other ME isoforms, we have examined the possible role of Lys346 by replacing it to alanine, serine or arginine. Our kinetic data indicate that the K346S mutant Enzyme displays a shift in its cofactor preference from NAD+ to NADP+ upon increasing kcat,NADP and decreasing Km,NADP. Furthermore, the cooperative binding of malate becomes less significant in human m-NAD-ME after mutation of Lys346. The h value for the wild-type is close to 2, but those of the K346 mutants are approximately 1.5. The K346 mutants can also be activated by fumarate and the cooperative effect can be abolished by fumarate, suggesting that the allosteric property is retained in these mutants. Our data strongly suggest that Lys346 in human m-NAD-ME is required for ATP inhibition. Mutation of Lys346 to Ser or Ala causes the Enzyme to be much less sensitive to ATP, similar to cytosolic NADP-dependent Malic Enzyme. Substitution of Lys to Arg did not change the isoform-specific inhibition of the Enzyme by ATP. The inhibition constants of ATP are increased for K346S and K346A, but are similar to those of the wild-type for K346R, suggesting that the positive charge rather than group specificity is required for binding affinity of ATP. Thus, ATP inhibition is proposed to be determined by the electrostatic potential involving the positive charge on the side chain of Lys346.

  • determinants of the dual cofactor specificity and substrate cooperativity of the human mitochondrial nad p dependent Malic Enzyme functional roles of glutamine 362
    Journal of Biological Chemistry, 2006
    Co-Authors: Juyi Hsieh, Gu-gang Chang, Guangyaw Liu, Huichih Hung
    Abstract:

    The human mitochondrial NAD(P)+-dependent Malic Enzyme (m-NAD-ME) is a Malic Enzyme isoform with dual cofactor specificity and substrate binding cooperativity. Previous kinetic studies have suggested that Lys362 in the pigeon cytosolic NADP+-dependent Malic Enzyme has remarkable effects on the binding of NADP+ to the Enzyme and on the catalytic power of the Enzyme (Kuo, C. C., Tsai, L. C., Chin, T. Y., Chang, G.-G., and Chou, W. Y. (2000) Biochem. Biophys. Res. Commun. 270, 821-825). In this study, we investigate the important role of Gln362 in the transformation of cofactor specificity from NAD+ to NADP+ in human m-NAD-ME. Our kinetic data clearly indicate that the Q362K mutant shifted its cofactor preference from NAD+ to NADP+. The Km(NADP) and kcat(NADP) values for this mutant were reduced by 4-6-fold and increased by 5-10-fold, respectively, compared with those for the wild-type Enzyme. Furthermore, up to a 2-fold reduction in Km(NADP)/Km(NAD) and elevation of kcat(NADP)/kcat(NAD) were observed for the Q362K Enzyme. Mutation of Gln362 to Ala or Asn did not shift its cofactor preference. The Km(NADP)/Km(NAD) and kcat(NADP)/kcat(NAD) values for Q362A and Q362N were comparable with those for the wild-type Enzyme. The ΔG values for Q362A and Q362N with either NAD+ or NADP+ were positive, indicating that substitution of Gln with Ala or Asn at position 362 brings about unfavorable cofactor binding at the active site and thus significantly reduces the catalytic efficiency. Our data also indicate that the cooperative binding of malate became insignificant in human m-NAD-ME upon mutation of Gln362 to Lys because the sigmoidal phenomenon appearing in the wild-type Enzyme was much less obvious that that in Q362K. Therefore, mutation of Gln362 to Lys in human m-NAD-ME alters its kinetic properties of cofactor preference, malate binding cooperativity, and allosteric regulation by fumarate. However, the other Gln362 mutants, Q362A and Q362N, have conserved malate binding cooperativity and NAD+ specificity. In this study, we provide clear evidence that the single mutation of Gln362 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.

Liang Tong - One of the best experts on this subject based on the ideXlab platform.

  • structural studies of the pigeon cytosolic nadp dependent Malic Enzyme
    Protein Science, 2009
    Co-Authors: Zhiru Yang, Gu-gang Chang, Hailong Zhang, Huichi Hung, Chenchin Kuo, Lichu Tsai, Hanna S Yuan, Weiyuan Chou, Liang Tong
    Abstract:

    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.

  • crystal structures of substrate complexes of Malic Enzyme and insights into the catalytic mechanism
    Structure, 2003
    Co-Authors: Xiao Tao, Zhiru Yang, Liang Tong
    Abstract:

    Abstract Malic Enzymes catalyze the oxidative decarboxylation of L -malate to pyruvate and CO 2 with the reduction of the NAD(P) + cofactor in the presence of divalent cations. We report the crystal structures at up to 2.1 A resolution of human mitochondrial NAD(P) + -dependent Malic Enzyme in different pentary complexes with the natural substrate malate or pyruvate, the dinucleotide cofactor NAD + or NADH, the divalent cation Mn 2+ , and the allosteric activator fumarate. Malate is bound deep in the active site, providing two ligands for the cation, and its C4 carboxylate group is out of plane with the C1-C2-C3 atoms, facilitating decarboxylation. The divalent cation is positioned optimally to catalyze the entire reaction. Lys183 is the general base for the oxidation step, extracting the proton from the C2 hydroxyl of malate. Tyr112-Lys183 functions as the general acid-base pair to catalyze the tautomerization of the enolpyruvate product from decarboxylation to pyruvate.

  • molecular mechanism for the regulation of human mitochondrial nad p dependent Malic Enzyme by atp and fumarate
    Structure, 2002
    Co-Authors: Zhiru Yang, Charles W Lanks, Liang Tong
    Abstract:

    The regulation of human mitochondrial NAD(P)+-dependent Malic Enzyme (m-NAD-ME) by ATP and fumarate may be crucial for the metabolism of glutamine for energy production in rapidly proliferating tissues and tumors. Here we report the crystal structure at 2.2 A resolution of m-NAD-ME in complex with ATP, Mn2+, tartronate, and fumarate. Our structural, kinetic, and mutagenesis studies reveal unexpectedly that ATP is an active-site inhibitor of the Enzyme, despite the presence of an exo binding site. The structure also reveals the allosteric binding site for fumarate in the dimer interface. Mutations in this binding site abolished the activating effects of fumarate. Comparison to the structure in the absence of fumarate indicates a possible molecular mechanism for the allosteric function of this compound.

  • Structure of a closed form of human Malic Enzyme and implications for catalytic mechanism
    Nature Structural Biology, 2000
    Co-Authors: Zhiru Yang, Daniel L. Floyd, Gerhard Loeber, Liang Tong
    Abstract:

    Malic Enzymes are widely distributed in nature and have many biological functions. The crystal structure of human mitochondrial NAD(P)^+-dependent Malic Enzyme in a quaternary complex with NAD^+, Mn^++ and oxalate has been determined at 2.2 Å resolution. The structures of the quaternary complex with NAD^+, Mg^++, tartronate or ketomalonate have been determined at 2.6 Å resolution. The structures show the Enzyme in a closed form in these complexes and reveal the binding modes of the cation and the inhibitors. The divalent cation is coordinated in an octahedral fashion by six ligating oxygens, two from the substrate/inhibitor, three from Glu 255, Asp 256 and Asp 279 of the Enzyme, and one from a water molecule. The structural information has significant implications for the catalytic mechanism of Malic Enzymes and identifies Tyr 112 and Lys 183 as possible catalytic residues. Changes in tetramer organization of the Enzyme are also observed in these complexes, which might be relevant for its cooperative behavior and allosteric control.