Mariel C Gerrard Wheeler

Rosario National University, Rosario, Santa Fe, Argentina

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Publications (12)44.83 Total impact

  • [show abstract] [hide abstract]
    ABSTRACT: C4 photosynthesis enables the capture of atmospheric CO2 and its concentration at the site of RuBisCO, thus counteracting the negative effects of low atmospheric levels of CO2 and high atmospheric levels of O2 (21 %) on photosynthesis. The evolution of this complex syndrome was a multistep process. It did not occur by simply recruiting pre-exiting components of the pathway from C3 ancestors which were already optimized for C4 function. Rather it involved modifications in the kinetics and regulatory properties of pre-existing isoforms of non-photosynthetic enzymes in C3 plants. Thus, biochemical studies aimed at elucidating the functional adaptations of these enzymes are central to the development of an integrative view of the C4 mechanism. In the present review, the most important biochemical approaches that we currently use to understand the evolution of the C4 isoforms of malic enzyme are summarized. It is expected that this information will help in the rational design of the best decarboxylation processes to provide CO2 for RuBisCO in engineering C3 species to perform C4 photosynthesis.
    Photosynthesis Research 07/2013; · 3.15 Impact Factor
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    ABSTRACT: Arabidopsis thaliana is a plant species that accumulates high levels of organic acids and uses them as carbon, energy and reducing power sources. Among the enzymes that metabolize these compounds, one of the most important ones is malic enzyme (ME). A. thaliana contains four malic enzymes (NADP-ME 1-4) to catalyze the reversible oxidative decarboxylation of malate in the presence of NADP. NADP-ME2 is the only one located in the cell cytosol of all Arabidopsis organs providing most of the total NADP-ME activity. In the present work, the regulation of this key enzyme by fumarate was investigated by kinetic assays, structural analysis and a site-directed mutagenesis approach. The final effect of this metabolite on NADP-ME2 forward activity not only depends on fumarate and substrate concentrations but also on the pH of the reaction medium. Fumarate produced an increase in NADP-ME2 activity by binding to an allosteric site. However at higher concentrations, fumarate caused a competitive inhibition, excluding the substrate malate from binding to the active site. The characterization of ME2-R115A mutant, which is not activated by fumarate, confirms this hypothesis. In addition, the reverse reaction (reductive carboxylation of pyruvate) is also modulated by fumarate, but in a different way. The results indicate pH-dependence of the fumarate modulation with opposite behavior on the two activities analyzed. Thereby, the coordinated action of fumarate over the direct and reverse reactions would allow a precise and specific modulation of the metabolic flux through this enzyme, leading to the synthesis or degradation of C(4) compounds under certain conditions. Thus, the physiological context might be exerting an accurate control of ME activity in planta, through changes in metabolite and substrate concentrations and cytosolic pH.
    Plant Molecular Biology 12/2012; · 3.52 Impact Factor
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    ABSTRACT: • While photosynthetic NADP-malic enzyme (NADP-ME) has a prominent role in the C(4) cycle, the biological function of nonphotosynthetic isoforms remains elusive. Here, we analysed the link between Arabidopsis thaliana cytosolic NADP-ME2 and the plant defence response. • Arabidopsis thaliana plants with wild-type and modified NADP-ME2 expression levels were analysed after elicitation with pathogen-associated molecular patterns (PAMPs) and during the interaction with the hemibiotrophic fungal pathogen Colletotrichum higginsianum. • Under normal growth conditions, the lack or gain of NADP-ME2 activity produced large changes in plant metabolite pool sizes without any effect on morphology or development. Total NADP-ME activity and NADP-ME2 transcript level were enhanced after PAMP treatment and pathogen infection. During infection with C. higginsianum, loss-of-function mutants of NADP-ME2 (nadp-me2) showed enhanced susceptibility. Transient apoplastic reactive oxygen species (ROS) production after elicitation and callose papilla formation after infection were dampened in nadp-me2. Late salicylic acid (SA)-dependent and SA-independent defence responses were not affected. • Taken together, our results indicate that NADP-ME2 is an important player in plant basal defence, where it appears to be involved in the generation of ROS. Moreover, NADP-ME2 was found to be dispensable for later defence responses.
    New Phytologist 04/2012; 195(1):189-202. · 6.74 Impact Factor
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    ABSTRACT: Arabidopsis mitochondria contain two NAD(+)-malic enzymes, NAD-ME1 and NAD-ME2. These proteins have similar affinity for their substrates but display opposite regulation by fumarate, which strongly stimulates NAD-ME1 but inhibits NAD-ME2 activity. Here, the interaction of NAD-ME1 and -2 with fumarate was investigated by kinetic approaches, urea denaturation assays and intrinsic fluorescence quenching, in the absence and presence of NAD(+). Fumarate inhibited NAD-ME2 at saturating, but not at low, levels of NAD(+), and it behaved as competitive inhibitor with respect to L-malate. In contrast, NAD-ME1 fumarate activation was higher at suboptimal NAD(+) concentrations. In the absence of cofactor, the fluorescence of both NAD-ME1 and -2 is quenched by fumarate. However, for NAD-ME2 the quenching arises from a collisional phenomenon, while in NAD-ME1 the fluorescence decay can be explained by a static process that involves fumarate binding to the protein. Furthermore, the residue Arg84 of NAD-ME1 is essential for fumarate binding, as the mutant protein R84A exhibits a collisional quenching by this metabolite. Together, the results indicate that the differential fumarate regulation of Arabidopsis NAD-MEs, which is further modulated by NAD(+) availability, is related to the gaining of an allosteric site for fumarate in NAD-ME1 and an active site-associated inhibition by this C(4)-organic acid in NAD-ME2.
    Biochimie 04/2012; 94(6):1421-30. · 3.14 Impact Factor
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    ABSTRACT: The Arabidopsis thaliana genome contains two genes encoding NAD-MEs [NAD-dependent malic enzymes; NAD-ME1 (TAIR accession number At4G13560) and NAD-ME2 (TAIR accession number At4G00570)]. The encoded proteins are localized to mitochondria and assemble as homo- and hetero- dimers in vitro and in vivo. In the present work, the kinetic mechanisms of NAD-ME1 and -ME2 homodimers and NAD-MEH (NAD-ME heterodimer) were studied as an approach to understand the contribution of these enzymes to plant physiology. Product-inhibition and substrate-analogue analyses indicated that NAD-ME2 follows a sequential ordered Bi-Ter mechanism, NAD being the leading substrate followed by L-malate. On the other hand, NAD-ME1 and NAD-MEH can bind both substrates randomly. However, NAD-ME1 shows a preferred route that involves the addition of NAD first. As a consequence of the kinetic mechanism, NAD-ME1 showed a partial inhibition by L-malate at low NAD concentrations. The analysis of a protein chimaeric for NAD-ME1 and -ME2 indicated that the first 176 amino acids are associated with the differences observed in the kinetic mechanisms of the enzymes. Furthermore, NAD-ME1, -ME2 and -MEH catalyse the reverse reaction (pyruvate reductive carboxylation) with very low catalytic activity, supporting the notion that these isoforms act only in L-malate oxidation in plant mitochondria. The different kinetic mechanism of each NAD-ME entity suggests that, for a metabolic condition in which the mitochondrial NAD level is low and the L-malate level is high, the activity of NAD-ME2 and/or -MEH would be preferred over that of NAD-ME1.
    Biochemical Journal 09/2010; 430(2):295-303. · 4.65 Impact Factor
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    ABSTRACT: The Arabidopsis thaliana genome contains four genes encoding NADP-malic enzymes (NADP-ME1-4). Two isoenzymes, NADP-ME2 and NADP-ME3, which are shown to be located in the cytosol, share a remarkably high degree of identity (90%). However, they display different expression patterns and show distinct kinetic properties, especially with regard to their regulation by effectors, in both the forward (malate oxidative decarboxylation) and reverse (pyruvate reductive carboxylation) reactions. In order to identify the domains in the primary structure that could be responsible for the regulatory differences, four chimeras between these isoenzymes were constructed and analysed. All chimeric versions exhibited the same native structures as the parental proteins. Analysis of the chimeras constructed indicated that the region from amino acid residue 303 to the C-terminal end of NADP-ME2 is critical for fumarate activation. However, the region flanked by amino acid residues 303 and 500 of NADP-ME3 is involved in the pH-dependent inhibition by high malate concentration. Furthermore, the N-terminal region of NADP-ME2 is necessary for the activation by succinate of the reverse reaction. Overall, the results show that NADP-ME2 and NADP-ME3 are able to distinguish and interact differently with similar C(4) acids as a result of minimal structural differences. Therefore, although the active sites of NADP-ME2 and NADP-ME3 are highly conserved, both isoenzymes acquire different allosteric sites, leading to the creation of proteins with unique regulatory mechanisms, probably best suited to the specific organ and developmental pattern of expression of each isoenzyme.
    FEBS Journal 10/2009; 276(19):5665-77. · 4.25 Impact Factor
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    ABSTRACT: Arabidopsis thaliana contains six genes encoding active malic enzymes (AtMEs). But, are all of them just involved in l-malate degradation? The presence of the six AtMEs can be easily attributed to genetic redundancy as none of the single knock-out mutants show a visible phenotype under normal conditions. However, the AtMEs display differential patterns of expression and well-distinct biochemical properties. In this regard, four AtMEs use NADP, three of which are cytosolic and one plastidic. While one cytosolic and the plastidic isoforms are constitutively expressed, the other cytosolic isoforms are exclusively found in roots, trichomes or pollen. The other two AtMEs use NAD, are localized in the mitochondria and are active as homo- and hetero-oligomers. Although AtMEs share a high degree of identity (up to 90%) they display different kinetic properties and metabolite regulation and some of the isoforms are active in the l-malate synthesis direction. Thus, the physiological context might also be controlling the functional specificity in planta, due to differences in metabolite concentrations in the compartments in which each AtME is expressed. As a whole, the divergent properties of the AtMEs allow us to propose that each ME fulfils an exclusive metabolic function in vivo. Moreover, due to the well-distinct properties of each AtME, the co-expression of some MEs in the same cellular compartment would not imply redundancy but represents specificity of function.
    Plant Science. 01/2009;
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    ABSTRACT: The Arabidopsis thaliana genome contains four NADP-malic enzymes genes (NADP-ME1-4). NADP-ME4 is localized to plastids whereas the other isoforms are cytosolic. NADP-ME2 and 4 are constitutively expressed, while NADP-ME1 is restricted to secondary roots and NADP-ME3 to trichomes and pollen. Although the four isoforms share remarkably high degree of identity (75-90%), recombinant NADP-ME1 through 4 show distinct kinetic properties, both in the forward (malate oxidative decarboxylation) and reverse (pyruvate reductive carboxylation) reactions. The four isoforms behave differently in terms of reversibility, with NADP-ME2 presenting the highest reverse catalytic efficiency. When analyzing the activity of each isoform in the presence of metabolic effectors, NADP-ME2 was the most highly regulated isoform, especially in its activation by certain effectors. Several metabolites modulate both the forward and reverse reactions, exhibiting dual effects in some cases. Therefore, pyruvate reductive carboxylation may be relevant in vivo, especially in some cellular compartments and conditions. In order to identify residues or segments of the NADP-ME primary structure that could be involved in the differences among the isoforms, NADP-ME2 mutants and deletions were analysed. The results obtained show that Arg115 is involved in fumarate activation, while the amino-terminal part is critical for aspartate and CoA activation, as well as for the reverse reaction. As a whole, these studies show that minimal changes in the primary structure are responsible for the different kinetic behaviour of each AtNADP-ME isoform. In this way, the co-expression of some isoforms in the same cellular compartment would not imply redundancy but represents specificity of function.
    Plant Molecular Biology 07/2008; 67(3):231-42. · 3.52 Impact Factor
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    ABSTRACT: The Arabidopsis (Arabidopsis thaliana) genome contains four genes encoding putative NADP-malic enzymes (MEs; AtNADP-ME1-ME4). NADP-ME4 is localized to plastids, whereas the other three isoforms do not possess any predicted organellar targeting sequence and are therefore expected to be cytosolic. The plant NADP-MEs can be classified into four groups: groups I and II comprising cytosolic and plastidic isoforms from dicots, respectively; group III containing isoforms from monocots; and group IV composed of both monocots and dicots, including AtNADP-ME1. AtNADP-MEs contained all conserved motifs common to plant NADP-MEs and the recombinant isozymes showed different kinetic and structural properties. NADP-ME2 exhibits the highest specific activity, while NADP-ME3 and NADP-ME4 present the highest catalytic efficiency for NADP and malate, respectively. NADP-ME4 exists in equilibrium of active dimers and tetramers, while the cytosolic counterparts are present as hexamers or octamers. Characterization of T-DNA insertion mutant and promoter activity studies indicates that NADP-ME2 is responsible for the major part of NADP-ME activity in mature tissues of Arabidopsis. Whereas NADP-ME2 and -ME4 are constitutively expressed, the expression of NADP-ME1 and NADP-ME3 is restricted by both developmental and cell-specific signals. These isoforms may play specific roles at particular developmental stages of the plant rather than being involved in primary metabolism.
    Plant physiology 10/2005; 139(1):39-51. · 6.56 Impact Factor
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    ABSTRACT: Malic enzymes catalyze the oxidative decarboxylation of l-malate to yield pyruvate, CO(2), and NAD(P)H in the presence of a bivalent metal ion. In plants, different isoforms of the NADP-malic enzyme (NADP-ME) are involved in a wide range of metabolic pathways. The C(4)-specific NADP-ME has evolved from C(3)-type malic enzymes to represent a unique and specialized form of NADP-ME as indicated by its particular kinetic and regulatory properties. In the present study, the mature C(4)-specific NADP-ME of maize was expressed in Escherichia coli. The recombinant enzyme has essentially the same physicochemical properties and K(m) for the substrates as those of the naturally occurring NADP-ME previously characterized. However, the k(cat) was almost 7-fold higher, which may suggest that the previously purified enzyme from maize leaves was partially inactive. The recombinant NADP-ME also has a very low intrinsic NAD-dependent activity. Five mutants of NADP-ME at the postulated putative NADP-binding site(s) (Gsite5V, Gsite2V, A392G, A387G, and R237L) were constructed by site-directed mutagenesis and purified to homogeneity. The participation of these residues in substrate binding and/or the catalytic reaction was inferred by kinetic measurements and circular dichroism and intrinsic fluorescence spectra. The results obtained were compared with a predicted three-dimensional model of maize C(4) NADP-ME based on crystallographic studies of related animal NAD(P)-MEs. The data presented here represent the first prokaryotic expression of a plant NADP-ME and reveals valuable insight regarding the participation of the mutated amino acids in the binding of substrates and/or catalysis.
    Journal of Biological Chemistry 05/2003; 278(16):13757-64. · 4.65 Impact Factor
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    [show abstract] [hide abstract]
    ABSTRACT: Malic enzymes catalyze the oxidative decarboxylation of l-malate to yield pyruvate, CO2, and NAD(P)H in the presence of a bivalent metal ion. In plants, different isoforms of the NADP-malic enzyme (NADP-ME) are involved in a wide range of metabolic pathways. The C4-specific NADP-ME has evolved from C3-type malic enzymes to represent a unique and specialized form of NADP-ME as indicated by its particular kinetic and regulatory properties. In the present study, the mature C4-specific NADP-ME of maize was expressed in Escherichia coli. The recombinant enzyme has essentially the same physicochemical properties andK m for the substrates as those of the naturally occurring NADP-ME previously characterized. However, thek cat was almost 7-fold higher, which may suggest that the previously purified enzyme from maize leaves was partially inactive. The recombinant NADP-ME also has a very low intrinsic NAD-dependent activity. Five mutants of NADP-ME at the postulated putative NADP-binding site(s) (Gsite5V, Gsite2V, A392G, A387G, and R237L) were constructed by site-directed mutagenesis and purified to homogeneity. The participation of these residues in substrate binding and/or the catalytic reaction was inferred by kinetic measurements and circular dichroism and intrinsic fluorescence spectra. The results obtained were compared with a predicted three-dimensional model of maize C4 NADP-ME based on crystallographic studies of related animal NAD(P)-MEs. The data presented here represent the first prokaryotic expression of a plant NADP-ME and reveals valuable insight regarding the participation of the mutated amino acids in the binding of substrates and/or catalysis.
    Journal of Biological Chemistry 04/2003; 278(16):13757-13764. · 4.65 Impact Factor
  • Source
    [show abstract] [hide abstract]
    ABSTRACT: Malic enzymes catalyze the oxidative decarboxylation of L-malate to yield pyruvate, CO2, and NAD(P)H in the presence of a bivalent metal ion. In plants, different isoforms of the NADP-malic enzyme (NADP-ME) are in- volved in a wide range of metabolic pathways. The C4- specific NADP-ME has evolved from C3-type malic en- zymes to represent a unique and specialized form of NADP-ME as indicated by its particular kinetic and reg- ulatory properties. In the present study, the mature C4- specific NADP-ME of maize was expressed in Esche- richia coli. The recombinant enzyme has essentially the same physicochemical properties and Km for the sub- strates as those of the naturally occurring NADP-ME previously characterized. However, the kcat was almost 7-fold higher, which may suggest that the previously purified enzyme from maize leaves was partially inac- tive. The recombinant NADP-ME also has a very low intrinsic NAD-dependent activity. Five mutants of NADP-ME at the postulated putative NADP-binding site(s) (Gsite5V, Gsite2V, A392G, A387G, and R237L) were constructed by site-directed mutagenesis and pu- rified to homogeneity. The participation of these resi- dues in substrate binding and/or the catalytic reaction was inferred by kinetic measurements and circular di- chroism and intrinsic fluorescence spectra. The results obtained were compared with a predicted three-dimen- sional model of maize C4 NADP-ME based on crystallo- graphic studies of related animal NAD(P)-MEs. The data presented here represent the first prokaryotic expres- sion of a plant NADP-ME and reveals valuable insight regarding the participation of the mutated amino acids in the binding of substrates and/or catalysis.

Publication Stats

133 Citations
51 Downloads
666 Views
44.83 Total Impact Points

Institutions

  • 2003–2013
    • Rosario National University
      • Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI-CONICET)
      Rosario, Santa Fe, Argentina
  • 2009–2010
    • University of Cologne
      • Botanical Institute
      Köln, North Rhine-Westphalia, Germany