RESUMEN
The cornerstone of carbon (C) and nitrogen (N) metabolic interactions - respiration - is presently not well understood in plant cells: the source of the key intermediate 2-oxoglutarate (2OG), to which reduced N is combined to yield glutamate and glutamine, remains somewhat unclear. We took advantage of combined mutations of NAD- and NADP-dependent isocitrate dehydrogenase activity and investigated the associated metabolic effects in Arabidopsis leaves (the major site of N assimilation in this genus), using metabolomics and (13)C-labelling techniques. We show that a substantial reduction in leaf isocitrate dehydrogenase activity did not lead to changes in the respiration efflux rate but respiratory metabolism was reorchestrated: 2OG production was supplemented by a metabolic bypass involving both lysine synthesis and degradation. Although the recycling of lysine has long been considered important in sustaining respiration, we show here that lysine neosynthesis itself participates in an alternative respiratory pathway. Lys metabolism thus contributes to explaining the metabolic flexibility of plant leaves and the effect (or the lack thereof) of respiratory mutations.
Asunto(s)
Arabidopsis/enzimología , Arabidopsis/genética , Isocitrato Deshidrogenasa/deficiencia , Lisina/biosíntesis , Mutación/genética , Aminoácidos/metabolismo , Isótopos de Carbono , Respiración de la Célula , Gases/metabolismo , Isocitrato Deshidrogenasa/metabolismo , Metaboloma , Metabolómica , FotosíntesisRESUMEN
d-Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the most abundant enzyme on Earth and is responsible for the fixation of atmospheric CO(2) into biomass. The reaction consists of incorporation of CO(2) and solvent H(2)O into d-ribulose 1,5-bisphosphate (RuBP) to yield 3-phospho-d-glycerate. The reaction involves several proton-dependent events: abstraction and protonation during enolization of RuBP and hydrolysis and reprotonation of the six-carbon reaction intermediate (carboxyketone). Although much is known about Rubisco structure and diversity, fundamental aspects of the reaction mechanism are poorly documented. How and when are protons exchanged among substrate, amino acid residues, and solvent water, and could alterations of proton exchange influence catalytic turnover? What is the energy profile of the reaction? To answer these questions, we measured catalytic rates and the (12)CO(2)/(13)CO(2) isotope effect in isotopic waters. We show that with increasing D(2)O content, the maximal carboxylation velocity (k(cat)(c)) decreased linearly and was 1.7 times lower in pure D(2)O. By contrast, the isotope effect on the apparent Michaelis constant for CO(2) (K(c)) was unity, suggesting that H/D exchange might have occurred with the solvent in early steps thereby slowing the overall catalysis. Calculations of kinetic commitments from observed isotope effects further indicate that (1) enolization and processing of the carboxyketone are similarly rate-limiting and (2) the tendency of the carboxyketone to go backward (decarboxylation) is likely exacerbated upon deuteration. Our results thus suggest that Rubisco catalysis is achieved by a rather equal distribution of energy barriers along the reaction.
Asunto(s)
Óxido de Deuterio/metabolismo , Ribulosa-Bifosfato Carboxilasa/metabolismo , Spinacia oleracea/enzimología , Dióxido de Carbono/metabolismo , Cinética , Hojas de la Planta/metabolismo , Ribulosafosfatos/metabolismo , Spinacia oleracea/metabolismo , TermodinámicaRESUMEN
The stable carbon isotope (13)C is used as a universal tracer in plant eco-physiology and studies of carbon exchange between vegetation and atmosphere. Photosynthesis fractionates against (13)CO(2) so that source sugars (photosynthates) are on average (13)C depleted by 20 compared with atmospheric CO(2). The carbon isotope distribution within sugars has been shown to be heterogeneous, with relatively (13)C-enriched and (13)C-depleted C-atom positions. The (13)C pattern within sugars is the cornerstone of (13)C distribution in plants, because all metabolites inherit the (13)C abundance in their specific precursor C-atom positions. However, the intramolecular isotope pattern in source leaf glucose and the isotope fractionation associated with key enzymes involved in sugar interconversions are currently unknown. To gain insight into these, we have analyzed the intramolecular isotope composition in source leaf transient starch, grain storage starch, and root storage sucrose and measured the site-specific isotope fractionation associated with the invertase (EC 3.2.1.26) and glucose isomerase (EC 5.3.1.5) reactions. When these data are integrated into a simple steady-state model of plant isotopic fluxes, the enzyme-dependent fractionations satisfactorily predict the observed intramolecular patterns. These results demonstrate that glucose and sucrose metabolism is the primary determinant of the (13)C abundance in source and sink tissue and is, therefore, of fundamental importance to the interpretation of plant isotopic signals.
Asunto(s)
Isótopos de Carbono/análisis , Hexosas/química , Plantas/química , Cromatografía Líquida de Alta Presión , Modelos Teóricos , Fotosíntesis , Hojas de la Planta/químicaRESUMEN
*Nitrogen assimilation in leaves requires primary NH(2) acceptors that, in turn, originate from primary carbon metabolism. Respiratory metabolism is believed to provide such acceptors (such as 2-oxoglutarate), so that day respiration is commonly seen as a cornerstone for nitrogen assimilation into glutamate in illuminated leaves. However, both glycolysis and day respiratory CO(2) evolution are known to be inhibited by light, thereby compromising the input of recent photosynthetic carbon for glutamate production. *In this study, we carried out isotopic labelling experiments with (13)CO(2) and (15)N-ammonium nitrate on detached leaves of rapeseed (Brassica napus), and performed (13)C- and (15)N-nuclear magnetic resonance analyses. *Our results indicated that the production of (13)C-glutamate and (13)C-glutamine under a (13)CO(2) atmosphere was very weak, whereas (13)C-glutamate and (13)C-glutamine appeared in both the subsequent dark period and the next light period under a (12)CO(2) atmosphere. Consistently, the analysis of heteronuclear ((13)C-(15)N) interactions within molecules indicated that most (15)N-glutamate and (15)N-glutamine molecules were not (13)C labelled after (13)C/(15)N double labelling. That is, recent carbon atoms (i.e. (13)C) were hardly incorporated into glutamate, but new glutamate molecules were synthesized, as evidenced by (15)N incorporation. *We conclude that the remobilization of night-stored molecules plays a significant role in providing 2-oxoglutarate for glutamate synthesis in illuminated rapeseed leaves, and therefore the natural day : night cycle seems critical for nitrogen assimilation.
Asunto(s)
Brassica napus/metabolismo , Dióxido de Carbono/metabolismo , Ácido Glutámico/metabolismo , Marcaje Isotópico/métodos , Luz , Nitrógeno/metabolismo , Hojas de la Planta/metabolismo , Brassica napus/efectos de la radiación , Isótopos de Carbono , Oscuridad , Espectroscopía de Resonancia Magnética , Isótopos de Nitrógeno , Fotosíntesis/efectos de la radiación , Hojas de la Planta/efectos de la radiación , Marcadores de SpinRESUMEN
The carboxylation kinetic (stable carbon) isotope effect was measured for purified d-ribulose-1,5-bisphosphate carboxylases/oxygenases (Rubiscos) with aqueous CO(2) as substrate by monitoring Rayleigh fractionation using membrane inlet mass spectrometry. This resulted in discriminations (Delta) of 27.4 +/- 0.9 per thousand for wild-type tobacco Rubisco, 22.2 +/- 2.1 per thousand for Rhodospirillum rubrum Rubisco, and 11.2 +/- 1.6 per thousand for a large subunit mutant of tobacco Rubisco in which Leu(335) is mutated to valine (L335V). These Delta values are consistent with the photosynthetic discrimination determined for wild-type tobacco and transplastomic tobacco lines that exclusively produce R. rubrum or L335V Rubisco. The Delta values are indicative of the potential evolutionary variability of Delta values for a range of Rubiscos from different species: Form I Rubisco from higher plants; prokaryotic Rubiscos, including Form II; and the L335V mutant. We explore the implications of these Delta values for the Rubisco catalytic mechanism and suggest that Rubiscos that are associated with a lower Delta value have a less product-like carboxylation transition state and/or allow a decarboxylation step that evolution has excluded in higher plants.
Asunto(s)
Proteínas Bacterianas/química , Nicotiana/enzimología , Proteínas de Plantas/química , Rhodospirillum rubrum/enzimología , Ribulosa-Bifosfato Carboxilasa/química , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dióxido de Carbono/química , Dióxido de Carbono/metabolismo , Isótopos de Carbono/química , Catálisis , Mutación Missense , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/enzimología , Plantas Modificadas Genéticamente/genética , Rhodospirillum rubrum/genética , Ribulosa-Bifosfato Carboxilasa/genética , Ribulosa-Bifosfato Carboxilasa/metabolismo , Especificidad de la Especie , Nicotiana/genéticaRESUMEN
The cornerstone of autotrophy, the CO(2)-fixing enzyme, d-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), is hamstrung by slow catalysis and confusion between CO(2) and O(2) as substrates, an "abominably perplexing" puzzle, in Darwin's parlance. Here we argue that these characteristics stem from difficulty in binding the featureless CO(2) molecule, which forces specificity for the gaseous substrate to be determined largely or completely in the transition state. We hypothesize that natural selection for greater CO(2)/O(2) specificity, in response to reducing atmospheric CO(2):O(2) ratios, has resulted in a transition state for CO(2) addition in which the CO(2) moiety closely resembles a carboxylate group. This maximizes the structural difference between the transition states for carboxylation and the competing oxygenation, allowing better differentiation between them. However, increasing structural similarity between the carboxylation transition state and its carboxyketone product exposes the carboxyketone to the strong binding required to stabilize the transition state and causes the carboxyketone intermediate to bind so tightly that its cleavage to products is slowed. We assert that all Rubiscos may be nearly perfectly adapted to the differing CO(2), O(2), and thermal conditions in their subcellular environments, optimizing this compromise between CO(2)/O(2) specificity and the maximum rate of catalytic turnover. Our hypothesis explains the feeble rate enhancement displayed by Rubisco in processing the exogenously supplied carboxyketone intermediate, compared with its nonenzymatic hydrolysis, and the positive correlation between CO(2)/O(2) specificity and (12)C/(13)C fractionation. It further predicts that, because a more product-like transition state is more ordered (decreased entropy), the effectiveness of this strategy will deteriorate with increasing temperature.