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Papers by Carlos Andreo

Research paper thumbnail of NADP-Malic Enzyme from the C3-C4 Intermediate Specie Flaveria Floridana

Photosynthesis: Mechanisms and Effects, 1998

C4 plants have fully differentiated mesophyll (MC) and bundle sheath cells (BSC) which cooperate ... more C4 plants have fully differentiated mesophyll (MC) and bundle sheath cells (BSC) which cooperate to fix CO2 by the C4 pathway. C4 plants are more efficient than C3 under some environmental conditions due to the higher CO2 concentration in BSC. C3-C4 intermediate species are thought to represent a stage in the evolutionary transition from the C3 to the C4 photosynthetic mechanism (1,2). MC of C3-C4 species function, as in C3 plants, fixing atmospheric CO2 through RuBisCO and generating C-2 compounds for the photosynthetic oxidation cycle. Two mechanisms are proposed to account for the low apparent photorespiration in these intermediate species. In one of them, which may be common to all intermediates, metabolites generated as a consequence of the RuBisCO oxygenase reaction in MC are metabolized in BSC and the CO2 released refixed by RuBisCO. In this way, reduced photorespiratory CO2 evolution occurs without the operation of a C4 cycle. In other class of intermediates, the operation of a limited C4 cycle between MC and BSC contributes to the further reduction of photorespiration. Flaveria floridana, a C3-C4 intermediate specie, was used in the present report to characterize different isoforms of NADP-malic enzyme (NADP-ME) and to evaluate their expression in different photosynthetic cell types. In previous studies, we detected three isoforms of the enzyme in various Flaveria intermediate species (3). One of them was found to be constitutively expressed in photosynthetic and non-photosynthetic tissues of the different species examined, while the other two isoforms were abundant only in photosynthetic tissues having partial or complete C4 photosynthesis. A correlation between these proteins, their function and the cDNAs already cloned (4) is necessary to understand the evolution of this protein among the different Flaveria species.

Research paper thumbnail of Sunflower metallothionein family characterisation. Study of the Zn(II)- and Cd(II)-binding abilities of the HaMT1 and HaMT2 isoforms

Journal of inorganic biochemistry, Jan 28, 2015

Plant metallothioneins (MTs) constitute a family of small Cys-rich proteins capable of coordinati... more Plant metallothioneins (MTs) constitute a family of small Cys-rich proteins capable of coordinating metal ions, significantly differing from microbial and animal MTs. They are divided into four subfamilies depending on the Cys pattern in their sequence. In this work, the MT system of the sunflower plant (Helianthus annuus) has been defined, with ten genes coding for MTs (HaMT) belonging to the four plant MT subfamilies; three HaMT1, four HaMT2, one HaMT3 and two HaMT4 isoforms. The gene expression pattern and capacity to confer metal resistance to yeast cells have been analysed for at least one member of each subfamily. The divalent metal ion-binding abilities of HaMT1-2 and HaMT2-1 (the isoforms encoded by the most abundantly expressed HaMT1 and HaMT2 isogenes) have been characterised, as HaMT3 and HaMT4 were previously studied. Those isoforms constitute an optimum material to study the effect of Cys number variability on their coordination abilities, as they exhibit additional Cys...

Research paper thumbnail of Research Report

Archives of Biochemistry and Biophysics, 1997

modification of a guard cell protein that is specifically implicated in stomatal movements. ᭧ 199... more modification of a guard cell protein that is specifically implicated in stomatal movements. ᭧ 1997 Plants regulate water loss and CO 2 gain by modu-Academic Press lating the aperture sizes of stomata that penetrate Key Words: phosphoenolpyruvate carboxylase; PEPC; the epidermis. Aperture size itself is increased by phosphorylation; fusicoccin; FC; abscisic acid; ABA; osmolyte accumulation and consequent turgor inguard cells; stomata. crease in the pair of guard cells that flank each stoma. Guard cell phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), which catalyzes the regulated step leading to malate synthesis, is crucial for charge and pH maintenance during osmolyte accumulation. Stomata in the leaf epidermis provide the major pathway for Regulation of this cytosolic enzyme by effectors is gas exchange between plants and their environment. As stowell documented, but additional regulation by postmata are nonselective, water vapor exits while CO 2 enters translational modification is predicted by the alterthrough open stomata. To effect a compromise between the conflicting requirements for CO 2 uptake and water conserva-ation of PEPC kinetics during stomatal opening tion, plants have evolved mechanisms to regulate stomatal-(FEBS Lett. 352, 45-48). In this study, we have invesaperture size. The regulation is implemented through changes tigated whether this alteration is associated with the in osmotic pressure of the pair of guard cells that flank each phosphorylation status of this enzyme. Using sonistoma. In brief, during stomatal opening, guard cells accumucated epidermal peels (''isolated'' guard cells) prelate solutes, primarily K / salts (1). Uptake of K / through chanloaded with 32 PO 4 , we induced stomatal opening and nels is driven by membrane hyperpolarization, which results guard cell malate accumulation by incubation with from H / extrusion by the plasmalemma H /-ATPase. As one 5 mM fusicoccin (FC). In corroboratory experiments, means to maintain cellular pH during proton extrusion, guard guard cells were incubated with the FC antagonist, cells synthesize and accumulate organic anions, predominantly 10 mM abscisic acid (ABA). The phosphorylation stamalate (2). That is, for each carboxylate accumulated, a proton tus of PEPC was assessed by immunoprecipitation, is released to the cytosol as compensation for proton extrusion. electrophoresis, immunoblotting, and autoradiogra-Overall, the decrease in guard cell solute potential causes osphy. PEPC was phosphorylated when stomata were motic water uptake. Thus, guard cells swell, distending their stimulated to open, and phosphorylation was lesswalls asymmetrically and widening the aperture. Stomatal cloened by incubation with ABA. Thus, we conclude that sure is essentially the reverse as guard cells shrink due to solute regulation of guard cell PEPC in vivo is multifaceted; and water dissipation. the effects of regulatory metabolites and the activa-Phosphoenolpyruvate (PEP) 3 carboxylase (PEPC, EC tion status of the enzyme are integrated to control 4.1.1.31) is a cytosolic enzyme that catalyzes the branch-point malate synthesis. These results, together with the costep in the malate-accumulation pathway in guard cells durincident alteration in the kinetics of the enzyme ing stomatal opening. This guard cell enzyme is regulated by (FEBS Lett. 352, 45-48), constitute the first unequivocytosolic pH and the allosteric effectors (e.g., 3-5), glc-6-P (an activator) and malate (an inhibitor). PEPC isoforms exist cal demonstration of regulatory posttranslational 345

Research paper thumbnail of Understanding C4 photosynthesis in Setaria by a proteomic and kinetic approach

bioRxiv, 2021

Plants performing C4 photosynthesis have a higher productivity per crop area related to an optimi... more Plants performing C4 photosynthesis have a higher productivity per crop area related to an optimized use of water and nutrients. This is achieved through a series of anatomical and biochemical features that allow the concentration of CO2 around RuBisCO. In C4 plants the photosynthetic reactions are distributed between two cell types, they initially fix the carbon to C4 acids within the mesophyll cells (M) and then transport these compounds to the bundle sheath cells (BS), where they are decarboxylated so that the resulting CO2 is incorporated into the Calvin cycle (CC). This work is focused on the comparative analysis of the proteins present in M and BS of Setaria viridis, a C4 model close relative of several major feed, fuel, and bioenergy grasses. The integration of kinetic and proteomic approaches agrees that the C4 compound malate is mainly decarboxylated in the chloroplasts of BS cells by NADP-malic enzyme (NADP-ME). Besides, NAD-malic enzyme (NAD-ME) located in the mitochondri...

Research paper thumbnail of The C4 cycle and beyond: diverse metabolic adaptations accompany dual-cell photosynthetic functions in Setaria

Journal of Experimental Botany, 2021

C4 photosynthesis is typically characterized by the spatial compartmentalization of the photosynt... more C4 photosynthesis is typically characterized by the spatial compartmentalization of the photosynthetic reactions into mesophyll (M) and bundle sheath (BS) cells. Initial carbon fixation within M cells gives rise to C4 acids, which are transported to the BS cells. There, C4 acids are decarboxylated so that the resulting CO2 is incorporated into the Calvin cycle. This work is focused on the study of Setaria viridis, a C4 model plant, closely related to several major feed and bioenergy grasses. First, we performed the heterologous expression and biochemical characterization of Setaria isoforms for chloroplastic NADP-malic enzyme (NADP-ME) and mitochondrial NAD-malic enzyme (NAD-ME). The kinetic parameters obtained agree with a major role for NADP-ME in the decarboxylation of the C4 acid malate in the chloroplasts of BS cells. In addition, mitochondria-located NAD-ME showed regulatory properties that could be important in the context of the operation of the C4 carbon shuttle. Secondly, ...

Research paper thumbnail of Analogues of NADP(+) as inhibitors and coenzymes for NADP(+) malic enzyme from maize leaves

Photosynthesis research, 1991

Structural analogues of the NADP(+) were studied as potential coenzymes and inhibitors for NADP(+... more Structural analogues of the NADP(+) were studied as potential coenzymes and inhibitors for NADP(+) dependent malic enzyme from Zea mays L. leaves. Results showed that 1, N(6)-etheno-nicotinamide adenine dinucleotide phosphate (∈ NADP(+)), 3-acetylpyridine-adenine dinucleotide phosphate (APADP(+)), nicotinamide-hypoxanthine dinucleotide phosphate (NHDP(+)) and β-nicotinamide adenine dinucleotide 2': 3'-cyclic monophosphate (2'3'NADPc(+)) act as alternate coenzymes for the enzyme and that there is little variation in the values of the Michaelis constants and only a threefold variation in Vmax for the five nucleotides. On the other hand, thionicotinamide-adenine dinucleotide phosphate (SNADP(+)), 3-aminopyridine-adenine dinucleotide phosphate (AADP(+)), adenosine 2'-monophosphate (2'AMP) and adenosine 2': 3'-cyclic monophosphate (2'3'AMPc) were competitive inhibitors with respect to NADP(+), while β-nicotinamide adenine dinucleotide 3'-phosph...

Research paper thumbnail of Functional dissection of Escherichia coli phosphotransacetylase structural domains and analysis of key compounds involved in activity regulation

FEBS Journal, 2010

Escherichia coli phosphotransacetylase (Pta) catalyzes the reversible interconversion of acetyl-C... more Escherichia coli phosphotransacetylase (Pta) catalyzes the reversible interconversion of acetyl-CoA and acetyl phosphate. Both compounds are critical in E. coli metabolism, and acetyl phosphate is also involved in the regulation of certain signal transduction pathways. Along with acetate kinase, Pta plays an important role in acetate production when E. coli grows on rich medium; alternatively, it is involved in acetate utilization at high acetate concentrations. E. coli Pta is composed of three different domains, but only the C-terminal one, called PTA_PTB, is specific for all Ptas. In the present work, the characterization of E. coli Pta and deletions from the N-terminal region were performed. E. coli Pta acetyl phosphate-forming and acetyl phosphate-consuming reactions display different maximum activities, and are differentially regulated by pyruvate and phosphoenolpyruvate. These compounds activate acetyl phosphate production, but inhibit acetyl-CoA production, thus playing a critical role in defining the rates of the two Pta reactions. The characterization of three truncated Ptas, which all display Pta activity, indicates that the substrate-binding site is located at the C-terminal PTA_PTB domain. However, the N-terminal P-loop NTPase domain is involved in expression of the maximal catalytic activity, stabilization of the hexameric native state, and Pta activity regulation by NADH, ATP, phosphoenolpyruvate, and pyruvate. The truncated protein Pta-F3 was able to complement the growth on acetate of an E. coli mutant defective in acetyl-CoA synthetase and Pta, indicating that, although not regulated by metabolites, the Pta C-terminal domain is active in vivo.

Research paper thumbnail of Kinetic Analysis of the Non-Phosphorylated, in Vitro Phosphorylated, and Phosphorylation-Site-Mutant (Asp8) Forms of Intact Recombinant C4 Phosphoenolpyruvate Carboxylase from Sorghum

European Journal of Biochemistry, 1995

Research paper thumbnail of Characteristics and Physiological Function of NADP-Malic Enzyme from Wheat

Plant and Cell Physiology, 1997

Kinetic and structural properties of NADP-malic en-zyme (NADP-ME, EC 1.1.1.40) purified from stem... more Kinetic and structural properties of NADP-malic en-zyme (NADP-ME, EC 1.1.1.40) purified from stems and roots of wheat (Triticum aestivum), along with the possible physiological role of the enzyme were examined. Enzyme purification from stems sequentially involved ...

Research paper thumbnail of An Engineered Change in the L-Malate Sensitivity of a Site-Directed Mutant of Sorghum Phosphoenolpyruvate Carboxylase: The Effect of Sequential Mutagenesis and S-Carboxymethylation at Position 8

Archives of Biochemistry and Biophysics, 1993

Research paper thumbnail of The Ionic Strength Changes the Quaternary Structure of Phosphoenolpyruvate Carboxylase from Maize Leaves

Progress in Photosynthesis Research, 1987

Research paper thumbnail of Authors

See next page for additional authors

Research paper thumbnail of Short Communication Inhibitory Effect of Shikimic Acid on PEP Carboxylase Activity

The inhibitory effect of shikimic acid on phosphoenol-pyruvate carboxylase (PEPC) from Amaranthus... more The inhibitory effect of shikimic acid on phosphoenol-pyruvate carboxylase (PEPC) from Amaranthus leaves was evaluated. Shikimic acid behaved as a competitive inhibitor vs. phosphoenolpyruvate (PEP). Kt for PEPC inhibition was 22/iM. Phenylalanine, tyrosine and tryptophan did not show any effect on PEPC activity. Key words: C4 plant — Flavonoid — Phosphoenolpyruvate carboxylase — Shikimic acid. The shikimic acid pathway leads to the biosynthesis of aromatic amino acids in plants, bacteria and fungi. In plants, this route is located to some extent, (if not entirely) in the stroma of chloroplasts, although some research groups report the existence of a cytoplasmic counterpart (Hrazdina and Jensen 1992). The first reaction of this path-way is the condensation of one molecule of erythrose 4-phosphate with one molecule of phosphoenolpyruvate cata-lyzed by deoxyarabino heptulosonate phosphate (DAHP) synthase, and shikimic acid is one of the intermediates. Most of the aromatic compounds sy...

Research paper thumbnail of Maize C4 NADP-Malic Enzyme EXPRESSION IN ESCHERICHIA COLI AND CHARACTERIZATION OF SITE-DIRECTED MUTANTS AT THE

Malic enzymes catalyze the oxidative decarboxylation of L-malate to yield pyruvate, CO2, and NAD(... more 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

Research paper thumbnail of 1 Running title: NADP-malic enzyme and Hsp70 association

Number: +54-341-4370044 Subject area: Proteins, enzymes and metabolism Environmental and stress r... more Number: +54-341-4370044 Subject area: Proteins, enzymes and metabolism Environmental and stress responses Number of black and white figures: 6 (Six) Number of color figures: 1 (One) Number of tables: 1 (One)

Research paper thumbnail of Chemical modification of thylakoid bound enzymes

Research paper thumbnail of Effects of hydroxamic acids isolated from gramineae on atp synthesis in chloroplasts

Research paper thumbnail of Identification of genes involved in the drought adaptation and recovery in Portulaca oleracea by differential display

Plant Physiology and Biochemistry, 2015

Portulaca oleracea is one of the richest plant sources of ω-3 and ω-6 fatty acids and other compo... more Portulaca oleracea is one of the richest plant sources of ω-3 and ω-6 fatty acids and other compounds potentially valuable for nutrition. It is broadly established in arid, semiarid and well-watered fields, thus making it a promising candidate for research on abiotic stress resistance mechanisms. It is capable of withstanding severe drought and then of recovering upon rehydration. Here, the adaptation to drought and the posterior recovery was evaluated at transcriptomic level by differential display validated by qRT-PCR. Of the 2279 transcript-derived fragments amplified, 202 presented differential expression. Ninety of them were successfully isolated and sequenced. Selected genes were tested against different abiotic stresses in P. oleracea and the behavior of their orthologous genes in Arabidopsis thaliana was also explored to seek for conserved response mechanisms. In drought adapted and in recovered plants changes in expression of many protein metabolism-, lipid metabolism- and stress-related genes were observed. Many genes with unknown function were detected, which also respond to other abiotic stresses. Some of them are also involved in the seed desiccation/imbibition process and thus would be of great interest for further research. The potential use of candidate genes to engineer drought tolerance improvement and recovery is discussed.

Research paper thumbnail of The ionic strength changes the quaternary structure of phosphoenolpyruvate carboxylase from maize leaves

Research paper thumbnail of His-containing plant metallothioneins: comparative study of divalent metal-ion binding by plant MT3 and MT4 isoforms

JBIC, J. Biol. Inorg. Chem., 2014

Research paper thumbnail of NADP-Malic Enzyme from the C3-C4 Intermediate Specie Flaveria Floridana

Photosynthesis: Mechanisms and Effects, 1998

C4 plants have fully differentiated mesophyll (MC) and bundle sheath cells (BSC) which cooperate ... more C4 plants have fully differentiated mesophyll (MC) and bundle sheath cells (BSC) which cooperate to fix CO2 by the C4 pathway. C4 plants are more efficient than C3 under some environmental conditions due to the higher CO2 concentration in BSC. C3-C4 intermediate species are thought to represent a stage in the evolutionary transition from the C3 to the C4 photosynthetic mechanism (1,2). MC of C3-C4 species function, as in C3 plants, fixing atmospheric CO2 through RuBisCO and generating C-2 compounds for the photosynthetic oxidation cycle. Two mechanisms are proposed to account for the low apparent photorespiration in these intermediate species. In one of them, which may be common to all intermediates, metabolites generated as a consequence of the RuBisCO oxygenase reaction in MC are metabolized in BSC and the CO2 released refixed by RuBisCO. In this way, reduced photorespiratory CO2 evolution occurs without the operation of a C4 cycle. In other class of intermediates, the operation of a limited C4 cycle between MC and BSC contributes to the further reduction of photorespiration. Flaveria floridana, a C3-C4 intermediate specie, was used in the present report to characterize different isoforms of NADP-malic enzyme (NADP-ME) and to evaluate their expression in different photosynthetic cell types. In previous studies, we detected three isoforms of the enzyme in various Flaveria intermediate species (3). One of them was found to be constitutively expressed in photosynthetic and non-photosynthetic tissues of the different species examined, while the other two isoforms were abundant only in photosynthetic tissues having partial or complete C4 photosynthesis. A correlation between these proteins, their function and the cDNAs already cloned (4) is necessary to understand the evolution of this protein among the different Flaveria species.

Research paper thumbnail of Sunflower metallothionein family characterisation. Study of the Zn(II)- and Cd(II)-binding abilities of the HaMT1 and HaMT2 isoforms

Journal of inorganic biochemistry, Jan 28, 2015

Plant metallothioneins (MTs) constitute a family of small Cys-rich proteins capable of coordinati... more Plant metallothioneins (MTs) constitute a family of small Cys-rich proteins capable of coordinating metal ions, significantly differing from microbial and animal MTs. They are divided into four subfamilies depending on the Cys pattern in their sequence. In this work, the MT system of the sunflower plant (Helianthus annuus) has been defined, with ten genes coding for MTs (HaMT) belonging to the four plant MT subfamilies; three HaMT1, four HaMT2, one HaMT3 and two HaMT4 isoforms. The gene expression pattern and capacity to confer metal resistance to yeast cells have been analysed for at least one member of each subfamily. The divalent metal ion-binding abilities of HaMT1-2 and HaMT2-1 (the isoforms encoded by the most abundantly expressed HaMT1 and HaMT2 isogenes) have been characterised, as HaMT3 and HaMT4 were previously studied. Those isoforms constitute an optimum material to study the effect of Cys number variability on their coordination abilities, as they exhibit additional Cys...

Research paper thumbnail of Research Report

Archives of Biochemistry and Biophysics, 1997

modification of a guard cell protein that is specifically implicated in stomatal movements. ᭧ 199... more modification of a guard cell protein that is specifically implicated in stomatal movements. ᭧ 1997 Plants regulate water loss and CO 2 gain by modu-Academic Press lating the aperture sizes of stomata that penetrate Key Words: phosphoenolpyruvate carboxylase; PEPC; the epidermis. Aperture size itself is increased by phosphorylation; fusicoccin; FC; abscisic acid; ABA; osmolyte accumulation and consequent turgor inguard cells; stomata. crease in the pair of guard cells that flank each stoma. Guard cell phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), which catalyzes the regulated step leading to malate synthesis, is crucial for charge and pH maintenance during osmolyte accumulation. Stomata in the leaf epidermis provide the major pathway for Regulation of this cytosolic enzyme by effectors is gas exchange between plants and their environment. As stowell documented, but additional regulation by postmata are nonselective, water vapor exits while CO 2 enters translational modification is predicted by the alterthrough open stomata. To effect a compromise between the conflicting requirements for CO 2 uptake and water conserva-ation of PEPC kinetics during stomatal opening tion, plants have evolved mechanisms to regulate stomatal-(FEBS Lett. 352, 45-48). In this study, we have invesaperture size. The regulation is implemented through changes tigated whether this alteration is associated with the in osmotic pressure of the pair of guard cells that flank each phosphorylation status of this enzyme. Using sonistoma. In brief, during stomatal opening, guard cells accumucated epidermal peels (''isolated'' guard cells) prelate solutes, primarily K / salts (1). Uptake of K / through chanloaded with 32 PO 4 , we induced stomatal opening and nels is driven by membrane hyperpolarization, which results guard cell malate accumulation by incubation with from H / extrusion by the plasmalemma H /-ATPase. As one 5 mM fusicoccin (FC). In corroboratory experiments, means to maintain cellular pH during proton extrusion, guard guard cells were incubated with the FC antagonist, cells synthesize and accumulate organic anions, predominantly 10 mM abscisic acid (ABA). The phosphorylation stamalate (2). That is, for each carboxylate accumulated, a proton tus of PEPC was assessed by immunoprecipitation, is released to the cytosol as compensation for proton extrusion. electrophoresis, immunoblotting, and autoradiogra-Overall, the decrease in guard cell solute potential causes osphy. PEPC was phosphorylated when stomata were motic water uptake. Thus, guard cells swell, distending their stimulated to open, and phosphorylation was lesswalls asymmetrically and widening the aperture. Stomatal cloened by incubation with ABA. Thus, we conclude that sure is essentially the reverse as guard cells shrink due to solute regulation of guard cell PEPC in vivo is multifaceted; and water dissipation. the effects of regulatory metabolites and the activa-Phosphoenolpyruvate (PEP) 3 carboxylase (PEPC, EC tion status of the enzyme are integrated to control 4.1.1.31) is a cytosolic enzyme that catalyzes the branch-point malate synthesis. These results, together with the costep in the malate-accumulation pathway in guard cells durincident alteration in the kinetics of the enzyme ing stomatal opening. This guard cell enzyme is regulated by (FEBS Lett. 352, 45-48), constitute the first unequivocytosolic pH and the allosteric effectors (e.g., 3-5), glc-6-P (an activator) and malate (an inhibitor). PEPC isoforms exist cal demonstration of regulatory posttranslational 345

Research paper thumbnail of Understanding C4 photosynthesis in Setaria by a proteomic and kinetic approach

bioRxiv, 2021

Plants performing C4 photosynthesis have a higher productivity per crop area related to an optimi... more Plants performing C4 photosynthesis have a higher productivity per crop area related to an optimized use of water and nutrients. This is achieved through a series of anatomical and biochemical features that allow the concentration of CO2 around RuBisCO. In C4 plants the photosynthetic reactions are distributed between two cell types, they initially fix the carbon to C4 acids within the mesophyll cells (M) and then transport these compounds to the bundle sheath cells (BS), where they are decarboxylated so that the resulting CO2 is incorporated into the Calvin cycle (CC). This work is focused on the comparative analysis of the proteins present in M and BS of Setaria viridis, a C4 model close relative of several major feed, fuel, and bioenergy grasses. The integration of kinetic and proteomic approaches agrees that the C4 compound malate is mainly decarboxylated in the chloroplasts of BS cells by NADP-malic enzyme (NADP-ME). Besides, NAD-malic enzyme (NAD-ME) located in the mitochondri...

Research paper thumbnail of The C4 cycle and beyond: diverse metabolic adaptations accompany dual-cell photosynthetic functions in Setaria

Journal of Experimental Botany, 2021

C4 photosynthesis is typically characterized by the spatial compartmentalization of the photosynt... more C4 photosynthesis is typically characterized by the spatial compartmentalization of the photosynthetic reactions into mesophyll (M) and bundle sheath (BS) cells. Initial carbon fixation within M cells gives rise to C4 acids, which are transported to the BS cells. There, C4 acids are decarboxylated so that the resulting CO2 is incorporated into the Calvin cycle. This work is focused on the study of Setaria viridis, a C4 model plant, closely related to several major feed and bioenergy grasses. First, we performed the heterologous expression and biochemical characterization of Setaria isoforms for chloroplastic NADP-malic enzyme (NADP-ME) and mitochondrial NAD-malic enzyme (NAD-ME). The kinetic parameters obtained agree with a major role for NADP-ME in the decarboxylation of the C4 acid malate in the chloroplasts of BS cells. In addition, mitochondria-located NAD-ME showed regulatory properties that could be important in the context of the operation of the C4 carbon shuttle. Secondly, ...

Research paper thumbnail of Analogues of NADP(+) as inhibitors and coenzymes for NADP(+) malic enzyme from maize leaves

Photosynthesis research, 1991

Structural analogues of the NADP(+) were studied as potential coenzymes and inhibitors for NADP(+... more Structural analogues of the NADP(+) were studied as potential coenzymes and inhibitors for NADP(+) dependent malic enzyme from Zea mays L. leaves. Results showed that 1, N(6)-etheno-nicotinamide adenine dinucleotide phosphate (∈ NADP(+)), 3-acetylpyridine-adenine dinucleotide phosphate (APADP(+)), nicotinamide-hypoxanthine dinucleotide phosphate (NHDP(+)) and β-nicotinamide adenine dinucleotide 2': 3'-cyclic monophosphate (2'3'NADPc(+)) act as alternate coenzymes for the enzyme and that there is little variation in the values of the Michaelis constants and only a threefold variation in Vmax for the five nucleotides. On the other hand, thionicotinamide-adenine dinucleotide phosphate (SNADP(+)), 3-aminopyridine-adenine dinucleotide phosphate (AADP(+)), adenosine 2'-monophosphate (2'AMP) and adenosine 2': 3'-cyclic monophosphate (2'3'AMPc) were competitive inhibitors with respect to NADP(+), while β-nicotinamide adenine dinucleotide 3'-phosph...

Research paper thumbnail of Functional dissection of Escherichia coli phosphotransacetylase structural domains and analysis of key compounds involved in activity regulation

FEBS Journal, 2010

Escherichia coli phosphotransacetylase (Pta) catalyzes the reversible interconversion of acetyl-C... more Escherichia coli phosphotransacetylase (Pta) catalyzes the reversible interconversion of acetyl-CoA and acetyl phosphate. Both compounds are critical in E. coli metabolism, and acetyl phosphate is also involved in the regulation of certain signal transduction pathways. Along with acetate kinase, Pta plays an important role in acetate production when E. coli grows on rich medium; alternatively, it is involved in acetate utilization at high acetate concentrations. E. coli Pta is composed of three different domains, but only the C-terminal one, called PTA_PTB, is specific for all Ptas. In the present work, the characterization of E. coli Pta and deletions from the N-terminal region were performed. E. coli Pta acetyl phosphate-forming and acetyl phosphate-consuming reactions display different maximum activities, and are differentially regulated by pyruvate and phosphoenolpyruvate. These compounds activate acetyl phosphate production, but inhibit acetyl-CoA production, thus playing a critical role in defining the rates of the two Pta reactions. The characterization of three truncated Ptas, which all display Pta activity, indicates that the substrate-binding site is located at the C-terminal PTA_PTB domain. However, the N-terminal P-loop NTPase domain is involved in expression of the maximal catalytic activity, stabilization of the hexameric native state, and Pta activity regulation by NADH, ATP, phosphoenolpyruvate, and pyruvate. The truncated protein Pta-F3 was able to complement the growth on acetate of an E. coli mutant defective in acetyl-CoA synthetase and Pta, indicating that, although not regulated by metabolites, the Pta C-terminal domain is active in vivo.

Research paper thumbnail of Kinetic Analysis of the Non-Phosphorylated, in Vitro Phosphorylated, and Phosphorylation-Site-Mutant (Asp8) Forms of Intact Recombinant C4 Phosphoenolpyruvate Carboxylase from Sorghum

European Journal of Biochemistry, 1995

Research paper thumbnail of Characteristics and Physiological Function of NADP-Malic Enzyme from Wheat

Plant and Cell Physiology, 1997

Kinetic and structural properties of NADP-malic en-zyme (NADP-ME, EC 1.1.1.40) purified from stem... more Kinetic and structural properties of NADP-malic en-zyme (NADP-ME, EC 1.1.1.40) purified from stems and roots of wheat (Triticum aestivum), along with the possible physiological role of the enzyme were examined. Enzyme purification from stems sequentially involved ...

Research paper thumbnail of An Engineered Change in the L-Malate Sensitivity of a Site-Directed Mutant of Sorghum Phosphoenolpyruvate Carboxylase: The Effect of Sequential Mutagenesis and S-Carboxymethylation at Position 8

Archives of Biochemistry and Biophysics, 1993

Research paper thumbnail of The Ionic Strength Changes the Quaternary Structure of Phosphoenolpyruvate Carboxylase from Maize Leaves

Progress in Photosynthesis Research, 1987

Research paper thumbnail of Authors

See next page for additional authors

Research paper thumbnail of Short Communication Inhibitory Effect of Shikimic Acid on PEP Carboxylase Activity

The inhibitory effect of shikimic acid on phosphoenol-pyruvate carboxylase (PEPC) from Amaranthus... more The inhibitory effect of shikimic acid on phosphoenol-pyruvate carboxylase (PEPC) from Amaranthus leaves was evaluated. Shikimic acid behaved as a competitive inhibitor vs. phosphoenolpyruvate (PEP). Kt for PEPC inhibition was 22/iM. Phenylalanine, tyrosine and tryptophan did not show any effect on PEPC activity. Key words: C4 plant — Flavonoid — Phosphoenolpyruvate carboxylase — Shikimic acid. The shikimic acid pathway leads to the biosynthesis of aromatic amino acids in plants, bacteria and fungi. In plants, this route is located to some extent, (if not entirely) in the stroma of chloroplasts, although some research groups report the existence of a cytoplasmic counterpart (Hrazdina and Jensen 1992). The first reaction of this path-way is the condensation of one molecule of erythrose 4-phosphate with one molecule of phosphoenolpyruvate cata-lyzed by deoxyarabino heptulosonate phosphate (DAHP) synthase, and shikimic acid is one of the intermediates. Most of the aromatic compounds sy...

Research paper thumbnail of Maize C4 NADP-Malic Enzyme EXPRESSION IN ESCHERICHIA COLI AND CHARACTERIZATION OF SITE-DIRECTED MUTANTS AT THE

Malic enzymes catalyze the oxidative decarboxylation of L-malate to yield pyruvate, CO2, and NAD(... more 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

Research paper thumbnail of 1 Running title: NADP-malic enzyme and Hsp70 association

Number: +54-341-4370044 Subject area: Proteins, enzymes and metabolism Environmental and stress r... more Number: +54-341-4370044 Subject area: Proteins, enzymes and metabolism Environmental and stress responses Number of black and white figures: 6 (Six) Number of color figures: 1 (One) Number of tables: 1 (One)

Research paper thumbnail of Chemical modification of thylakoid bound enzymes

Research paper thumbnail of Effects of hydroxamic acids isolated from gramineae on atp synthesis in chloroplasts

Research paper thumbnail of Identification of genes involved in the drought adaptation and recovery in Portulaca oleracea by differential display

Plant Physiology and Biochemistry, 2015

Portulaca oleracea is one of the richest plant sources of ω-3 and ω-6 fatty acids and other compo... more Portulaca oleracea is one of the richest plant sources of ω-3 and ω-6 fatty acids and other compounds potentially valuable for nutrition. It is broadly established in arid, semiarid and well-watered fields, thus making it a promising candidate for research on abiotic stress resistance mechanisms. It is capable of withstanding severe drought and then of recovering upon rehydration. Here, the adaptation to drought and the posterior recovery was evaluated at transcriptomic level by differential display validated by qRT-PCR. Of the 2279 transcript-derived fragments amplified, 202 presented differential expression. Ninety of them were successfully isolated and sequenced. Selected genes were tested against different abiotic stresses in P. oleracea and the behavior of their orthologous genes in Arabidopsis thaliana was also explored to seek for conserved response mechanisms. In drought adapted and in recovered plants changes in expression of many protein metabolism-, lipid metabolism- and stress-related genes were observed. Many genes with unknown function were detected, which also respond to other abiotic stresses. Some of them are also involved in the seed desiccation/imbibition process and thus would be of great interest for further research. The potential use of candidate genes to engineer drought tolerance improvement and recovery is discussed.

Research paper thumbnail of The ionic strength changes the quaternary structure of phosphoenolpyruvate carboxylase from maize leaves

Research paper thumbnail of His-containing plant metallothioneins: comparative study of divalent metal-ion binding by plant MT3 and MT4 isoforms

JBIC, J. Biol. Inorg. Chem., 2014