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.
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...
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
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...
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, ...
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...
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.
Steady-state kinetic analyses were performed on the non-phosphorylated, in vitro phosphorylated a... more Steady-state kinetic analyses were performed on the non-phosphorylated, in vitro phosphorylated and phosphorylation-site mutant (Ser8→Asp) forms of purified recombinant sorghum C 4 phosphoenolpyruvate (P-pyruvate) carboxylase (EC 4.1.1.3 1) containing an intact N-terminus. Significant differences in certain kinetic parameters were observed between these three enzyme forms when activity was assayed at a suboptimal but near-physiological pH (7.3), but not at optimal pH (8.0). Most notably, at pH 7.3 the apparent K i for the negative allosteric effector l-malate was 0.17 mM, 1.2 mM and 0.45 mM while the apparent K a for the positive allosteric effector glucose 6-phosphate (Glc6P) at 1mM P-pyruvate was 1.3 mM, 0.28 mM and 0.45 mM for the dephosphorylated, phosphorylated and mutant forms of the enzyme, respectively. These and related kinetic analyses at pH 7.3 show that phosphorylation of C 4 P-pyruvate carboxylase near its N-terminus has a relatively minor effect on V and K m (total P-pyruvate) but has a dramatic effect on the extent of activation by Glc6P, type of inhibition by l-malate and, most especially, K a (Glc6P) and K i (l-malate). Thus, regulatory phosphorylation profoundly influences the interactive allosteric properties of this cytosolic C 4 -photosynthesis enzyme.
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 ...
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...
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
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)
Two hydroxamic acids isolated from maize extracts, 2,4-dihydroxy-7methoxy-1,4-(2H)-benzoxazin-3(4... more Two hydroxamic acids isolated from maize extracts, 2,4-dihydroxy-7methoxy-1,4-(2H)-benzoxazin-3(4H)-one (DIMBOA) and the 2-O-.8-D-glucopyranoside of DIMBOA, inhibit photophosphorylation by spinach chlo- roplasts. Both cyclic and noncycfic photophosphorylations were inhibited to the same extent. The concentrations producing 50% inhibition for DIMBOA and its glucoside were about 1 and 4 millimolar, respectively. These compounds inhibit coupled electron transport but do not affect basal or uncoupled electron transport. Both acids inhibit the ATPase activities of membrane-bound coupling factor 1 (CF1) and of purified CF1. On the basis of these results, it is concluded that DIMBOA and its glucoside act as energy transfer inhibitors of photophosphorylation.
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.
Metallothioneins (MTs) are a superfamily of Cys-rich, low-molecular weight metalloproteins that b... more Metallothioneins (MTs) are a superfamily of Cys-rich, low-molecular weight metalloproteins that bind heavy metal ions. These cytosolic metallopeptides, which exist in most living organisms, are thought to be involved in metal homeostasis, metal detoxification, and oxidative stress protection. In this work, we characterise the Zn(II)and Cd(II)-binding abilities of plant type 3 and type 4 MTs identified in soybean and sunflower, both of them being His-containing peptides. The recombinant metal-MT complexes synthesised in Zn(II) or Cd(II)-enriched Escherichia coli cultures have been analysed by ESI-MS, and CD, ICP-AES, and UV spectroscopies. His-to-Ala type 3 MT mutants have also been constructed and synthesised for the study of the role of His in divalent metal ion coordination. The results show comparable divalent metal-binding capacities for the MTs of type 3, and suggest, for the first time, the participation of their conserved C-term His residues in metal binding. Interesting features for the Zn(II)-binding abilities of type 4 MTs are also reported, as their variable His content may be considered crucial for their biological performance.
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.
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...
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
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...
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, ...
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...
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.
Steady-state kinetic analyses were performed on the non-phosphorylated, in vitro phosphorylated a... more Steady-state kinetic analyses were performed on the non-phosphorylated, in vitro phosphorylated and phosphorylation-site mutant (Ser8→Asp) forms of purified recombinant sorghum C 4 phosphoenolpyruvate (P-pyruvate) carboxylase (EC 4.1.1.3 1) containing an intact N-terminus. Significant differences in certain kinetic parameters were observed between these three enzyme forms when activity was assayed at a suboptimal but near-physiological pH (7.3), but not at optimal pH (8.0). Most notably, at pH 7.3 the apparent K i for the negative allosteric effector l-malate was 0.17 mM, 1.2 mM and 0.45 mM while the apparent K a for the positive allosteric effector glucose 6-phosphate (Glc6P) at 1mM P-pyruvate was 1.3 mM, 0.28 mM and 0.45 mM for the dephosphorylated, phosphorylated and mutant forms of the enzyme, respectively. These and related kinetic analyses at pH 7.3 show that phosphorylation of C 4 P-pyruvate carboxylase near its N-terminus has a relatively minor effect on V and K m (total P-pyruvate) but has a dramatic effect on the extent of activation by Glc6P, type of inhibition by l-malate and, most especially, K a (Glc6P) and K i (l-malate). Thus, regulatory phosphorylation profoundly influences the interactive allosteric properties of this cytosolic C 4 -photosynthesis enzyme.
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 ...
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...
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
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)
Two hydroxamic acids isolated from maize extracts, 2,4-dihydroxy-7methoxy-1,4-(2H)-benzoxazin-3(4... more Two hydroxamic acids isolated from maize extracts, 2,4-dihydroxy-7methoxy-1,4-(2H)-benzoxazin-3(4H)-one (DIMBOA) and the 2-O-.8-D-glucopyranoside of DIMBOA, inhibit photophosphorylation by spinach chlo- roplasts. Both cyclic and noncycfic photophosphorylations were inhibited to the same extent. The concentrations producing 50% inhibition for DIMBOA and its glucoside were about 1 and 4 millimolar, respectively. These compounds inhibit coupled electron transport but do not affect basal or uncoupled electron transport. Both acids inhibit the ATPase activities of membrane-bound coupling factor 1 (CF1) and of purified CF1. On the basis of these results, it is concluded that DIMBOA and its glucoside act as energy transfer inhibitors of photophosphorylation.
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.
Metallothioneins (MTs) are a superfamily of Cys-rich, low-molecular weight metalloproteins that b... more Metallothioneins (MTs) are a superfamily of Cys-rich, low-molecular weight metalloproteins that bind heavy metal ions. These cytosolic metallopeptides, which exist in most living organisms, are thought to be involved in metal homeostasis, metal detoxification, and oxidative stress protection. In this work, we characterise the Zn(II)and Cd(II)-binding abilities of plant type 3 and type 4 MTs identified in soybean and sunflower, both of them being His-containing peptides. The recombinant metal-MT complexes synthesised in Zn(II) or Cd(II)-enriched Escherichia coli cultures have been analysed by ESI-MS, and CD, ICP-AES, and UV spectroscopies. His-to-Ala type 3 MT mutants have also been constructed and synthesised for the study of the role of His in divalent metal ion coordination. The results show comparable divalent metal-binding capacities for the MTs of type 3, and suggest, for the first time, the participation of their conserved C-term His residues in metal binding. Interesting features for the Zn(II)-binding abilities of type 4 MTs are also reported, as their variable His content may be considered crucial for their biological performance.
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Papers by Carlos Andreo