RESUMO
In plants, the last step in the biosynthesis of the osmoprotectant glycine betaine (GB) is the NAD(+)-dependent oxidation of betaine aldehyde (BAL) catalysed by some aldehyde dehydrogenase (ALDH) 10 enzymes that exhibit betaine aldehyde dehydrogenase (BADH) activity. Given the irreversibility of the reaction, the short-term regulation of these enzymes is of great physiological relevance to avoid adverse decreases in the NAD(+):NADH ratio. In the present study, we report that the Spinacia oleracea BADH (SoBADH) is reversibly and partially inactivated by BAL in the absence of NAD(+)in a time- and concentration-dependent mode. Crystallographic evidence indicates that the non-essential Cys(450)(SoBADH numbering) forms a thiohemiacetal with BAL, totally blocking the productive binding of the aldehyde. It is of interest that, in contrast to Cys(450), the catalytic cysteine (Cys(291)) did not react with BAL in the absence of NAD(+) The trimethylammonium group of BAL binds in the same position in the inactivating or productive modes. Accordingly, BAL does not inactivate the C(450)SSoBADH mutant and the degree of inactivation of the A(441)I and A(441)C mutants corresponds to their very different abilities to bind the trimethylammonium group. Cys(450)and the neighbouring residues that participate in stabilizing the thiohemiacetal are strictly conserved in plant ALDH10 enzymes with proven or predicted BADH activity, suggesting that inactivation by BAL is their common feature. Under osmotic stress conditions, this novel partial and reversible covalent regulatory mechanism may contribute to preventing NAD(+)exhaustion, while still permitting the synthesis of high amounts of GB and avoiding the accumulation of the toxic BAL.
Assuntos
Betaína-Aldeído Desidrogenase/química , Betaína/análogos & derivados , Mutação de Sentido Incorreto , Proteínas de Plantas/química , Spinacia oleracea/enzimologia , Substituição de Aminoácidos , Betaína/química , Betaína-Aldeído Desidrogenase/genética , Domínio Catalítico , Cristalografia por Raios X , Ativação Enzimática , Proteínas de Plantas/genética , Spinacia oleracea/genéticaRESUMO
In the search for natural inhibitors of plant growth, we investigate the mechanism of action of the natural furoquinoline alkaloids isolated from Balfourodendron riedelianum (Rutaceae): evolitrine (1), kokusaginine (2), γ-fagarine (3), skimmianine (4) and maculosidine (5) on the photosynthesis light reactions. Their effect on the electron transport chain on thylakoids was analyzed. Alkaloids 1, 2, 4 and 5 inhibited ATP synthesis, basal, phosphorylating and uncoupled electron transport acting as Hill reaction inhibitors on spinach chloroplasts. Alkaloid 3 was not active. The inhibition and interaction site of alkaloids 1, 2, 4 and 5 on the non-cyclic electron transport chain was studied by polarography and fluorescence of the chlorophyll a (Chl a). The results indicate that the target for 1 was localized on the donor and acceptor side of PS II. In addition alkaloids 2 and 5 affect the PS I electron acceptors on leaf discs.
Assuntos
Alcaloides/isolamento & purificação , Alcaloides/farmacologia , Cloroplastos/metabolismo , Fotossíntese/efeitos dos fármacos , Quinolinas/química , Rutaceae/química , Spinacia oleracea/citologia , Trifosfato de Adenosina/biossíntese , Alcaloides/química , Sítios de Ligação , Clorofila/metabolismo , Clorofila A , Cloroplastos/efeitos dos fármacos , Transporte de Elétrons/efeitos dos fármacos , Complexo de Proteína do Fotossistema II/metabolismo , Folhas de Planta/citologia , Folhas de Planta/enzimologia , Folhas de Planta/metabolismo , Spinacia oleracea/enzimologia , Spinacia oleracea/metabolismo , Tilacoides/efeitos dos fármacos , Tilacoides/metabolismoRESUMO
Plant Aldehyde Dehydrogenase10 (ALDH10) enzymes catalyze the oxidation of ω-primary or ω-quaternary aminoaldehydes, but, intriguingly, only some of them, such as the spinach (Spinacia oleracea) betaine aldehyde dehydrogenase (SoBADH), efficiently oxidize betaine aldehyde (BAL) forming the osmoprotectant glycine betaine (GB), which confers tolerance to osmotic stress. The crystal structure of SoBADH reported here shows tyrosine (Tyr)-160, tryptophan (Trp)-167, Trp-285, and Trp-456 in an arrangement suitable for cation-π interactions with the trimethylammonium group of BAL. Mutation of these residues to alanine (Ala) resulted in significant K(m)(BAL) increases and V(max)/K(m)(BAL) decreases, particularly in the Y160A mutant. Tyr-160 and Trp-456, strictly conserved in plant ALDH10s, form a pocket where the bulky trimethylammonium group binds. This space is reduced in ALDH10s with low BADH activity, because an isoleucine (Ile) pushes the Trp against the Tyr. Those with high BADH activity instead have Ala (Ala-441 in SoBADH) or cysteine, which allow enough room for binding of BAL. Accordingly, the mutation A441I decreased the V(max)/K(m)(BAL) of SoBADH approximately 200 times, while the mutation A441C had no effect. The kinetics with other ω-aminoaldehydes were not affected in the A441I or A441C mutant, demonstrating that the existence of an Ile in the second sphere of interaction of the aldehyde is critical for discriminating against BAL in some plant ALDH10s. A survey of the known sequences indicates that plants have two ALDH10 isoenzymes: those known to be GB accumulators have a high-BAL-affinity isoenzyme with Ala or cysteine in this critical position, while non GB accumulators have low-BAL-affinity isoenzymes containing Ile. Therefore, BADH activity appears to restrict GB synthesis in non-GB-accumulator plants.
Assuntos
Aminoácidos/metabolismo , Betaína-Aldeído Desidrogenase/metabolismo , Betaína/análogos & derivados , Spinacia oleracea/enzimologia , Aminoácidos Aromáticos/metabolismo , Betaína/química , Betaína/metabolismo , Betaína-Aldeído Desidrogenase/química , Sítios de Ligação , Isoenzimas/química , Isoenzimas/metabolismo , Cinética , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Ligação Proteica , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Plant chloroplasts are particularly threatened by free radical attack. We incubated purified soluble spinach chloroplast F(0)F(1) (CF(0)F(1), EC 3.6.3.34) with an Fe(2+)/H(2)O(2)/ascorbate system, and about 60% inactivation of the ATPase activity was reached after 60 min. Inactivation was not prevented by omission of H(2)O(2), by addition of catalase or superoxide dismutase, nor by the scavengers mannitol, DMSO, or BHT. No evidence for enzyme fragmentation or oligomerization was detected by SDS-PAGE. The chloroplast ATP synthase is resistant to attack by the reactive oxygen species commonly found at the chloroplast level. DTT in the medium completely prevented the inhibition, and its addition after the inhibition partially recovered the activity of the enzyme. CF(0)F(1) thiol residues were lost upon oxidation. The rate of thiol modification was faster than the rate of enzyme inactivation, suggesting that the thiol residues accounting for the inhibition may be hindered. Enzyme previously oxidized by iodobenzoate was not further inhibited by the oxidative system. The production of ascorbyl radical was identified by EPR and is possibly related to CF(0)F(1) inactivation. It is thus suggested that the ascorbyl radical, which accumulates under plant stress, might regulate CF(0)F(1).
Assuntos
Ácido Ascórbico/farmacologia , ATPases de Cloroplastos Translocadoras de Prótons/metabolismo , Peróxido de Hidrogênio/farmacologia , Spinacia oleracea/enzimologia , Ácido Ascórbico/química , ATPases de Cloroplastos Translocadoras de Prótons/antagonistas & inibidores , ATPases de Cloroplastos Translocadoras de Prótons/genética , Desferroxamina/farmacologia , Ditiotreitol/farmacologia , Ácido Edético/farmacologia , Eletroforese em Gel de Poliacrilamida , Radicais Livres/farmacologia , Concentração de Íons de Hidrogênio , Cinética , Oxirredução/efeitos dos fármacos , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismoRESUMO
Chloroplast ATP-synthase is an H(+)/ATP-driven rotary motor in which a hydrophobic multi-subunit assemblage rotates within a hydrophilic stator, and subunit interactions dictate alternate-site catalysis. To explore the relevance of these interactions for catalysis we use hydrostatic pressure to induce conformational changes and/or subunit dissociation, and the resulting changes in the ATPase activity and oligomer structure are evaluated. Under moderate hydrostatic pressure (up to 60-80 MPa), ATPase activity is increased by 1.5-fold. This is not related to an increase in the affinity for ATP, but seems to correlate with an enhanced turnover induced by pressure, and an activation volume for the ATPase reaction of -23.7 ml/mol. Higher pressure (up to 200 MPa) leads to dissociation of the enzyme, as shown by enzyme inactivation, increased binding of 8-anilinonaphthalene-1-sulfonate (ANS) to hydrophobic regions, and labeling of specific Cys residues on the beta and alpha subunits by N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylene-4-diamine (IAEDANS). Compression-decompression cycles (between 0.1 and 200 MPa) inactivate CF(0)F(1) in a concentration-dependent manner, although after decompression no enzyme subunit is retained on a Sephadex-G-50 centrifuge column or is further labeled by IAEDANS. It is proposed that moderate hydrostatic pressures induce elastic compression of CF(0)F(1), leading to enhanced turnover. High pressure dissociation impairs the contacts needed for rotational catalysis.
Assuntos
ATPases de Cloroplastos Translocadoras de Prótons/metabolismo , Trifosfato de Adenosina/metabolismo , ATPases de Cloroplastos Translocadoras de Prótons/efeitos dos fármacos , ATPases de Cloroplastos Translocadoras de Prótons/isolamento & purificação , Etanol/farmacologia , Fluorescência , Hidrólise , Pressão Hidrostática , Subunidades Proteicas/isolamento & purificação , Subunidades Proteicas/metabolismo , Spinacia oleracea/enzimologia , Coloração e RotulagemRESUMO
Soluble purified CF(0)F(1) from chloroplasts was either oxidized or reduced and then incubated with [alpha-(32)P]ATP in the presence or in the absence of Mg(2+). Depending on the conditions of incubation, the enzyme showed different tight-nucleotide binding sites. In the presence of EDTA, two sites bind [alpha-(32)P]ATP from the reaction medium at different rates. Both sites promote ATP hydrolysis, since equimolar amounts of [alpha-(32)P]ATP and [alpha-(32)P]ADP are bound to the enzyme. In the presence of Mg(2+), only one site appears during the first hour of incubation, with characteristics similar to those described in the absence of Mg(2+). However, after this time a third site appears also permitting binding of ATP from the reaction medium, but in this case the bound ATP is not hydrolyzed. Covalent derivatization by 2-azido-[alpha-(32)P]ATP was used to distinguish between catalytic and noncatalytic sites. In the presence of Mg(2+), there are at least three distinct nucleotide binding sites that bind nucleotide tightly from the reaction medium: two of them are catalytic and one is noncatalytic.
Assuntos
Cloroplastos/enzimologia , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/metabolismo , Spinacia oleracea/enzimologia , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Domínio Catalítico , Cinética , ATPases Translocadoras de Prótons/isolamento & purificaçãoRESUMO
Sucrose-phosphate synthase (SPS, EC 2.4.1.14) biochemical properties and peptide composition have been analyzed in rice leaf seedlings. SPS was purified using DEAE-Sephacel chromatography, gel filtration on Sepharose 6B and anion exchange chromatography on Mono Q. At this stage two enzyme forms (SPS-I and -II) were separated. SPS-II was purified 90-fold; however, SPS-I presented a lower specific activity regarding the previous purification step and an unstable activity. Both enzyme forms had similar apparent Km values for Fru-6P but the SPS-I Km for UDP-Glc was ca. 10-fold higher than the SPS-II one. In addition, they differentiate in the capacity of being modulated by Glc-6-P and Pi: while SPS-II activity was inhibited by Pi and activated by Glc-6-P, SPS-I was not affected by either effectors. A native molecular mass of ca. 420 kDa was found by gel filtration. In SPS expression analysis using leaf rice and wheat germ SPS antibodies, a 116 kDa polypeptide was revealed in rice leaf extracts and no polypeptide was immunoactive in rice roots.
Assuntos
Glucosiltransferases/isolamento & purificação , Isoenzimas/isolamento & purificação , Oryza/enzimologia , Glucosiltransferases/química , Glucosiltransferases/metabolismo , Immunoblotting , Isoenzimas/química , Isoenzimas/metabolismo , Folhas de Planta/enzimologia , Plantas Tóxicas , Spinacia oleracea/enzimologia , Nicotiana/enzimologia , Triticum/enzimologia , Zea mays/enzimologiaRESUMO
The cleavage of disulfide bonds is the major modification of chloroplast fructose-1,6-bisphosphatase when the light-mediated ferredoxin-thioredoxin system enhances the activity of the enzyme. In vitro, only thiol-bearing compounds are functional in the stimulation of fructose 1,6-bisphosphate hydrolysis. This investigation was undertaken to determine the effectivity of other reductants for enhancing the catalytic capacity. In the presence of 1 mM fructose 1,6-bisphosphate and 0.1 mM Ca2+, the five-fold activation triggered by 3.5 mM tributylphosphine is further potentiated by 15% (v/v) 2-propanol. When the enzyme is incubated in the presence of 0.15 M sodium trichloroacetate in place of the cosolvent, NaH4B initially stimulates the activity but subsequently causes the inactivation of the enzyme. A model developed to analyze this dual effect suggests that the concerted action of fructose 1,6-bisphosphate, Ca2+ and trichloroacetate yields an enzyme form that is slightly activable by reduction (t0.5 = 28 min.). However, chloroplast fructose-1,6-bisphosphatase becomes highly sensitive to trichloroacetate inactivation (t0.5 = 5 min.) when NaH4B reduces fructose 1,6-bisphosphate. Hence, the thiol/disulfide exchange constitutes a particular case of reductive mechanisms that stimulate the activity of chloroplast fructose-1,6-bisphosphatase.