RESUMEN
The stroma of higher plant chloroplasts contains two thioredoxins (Trx) with different specificity for the reduction of protein disulfide bonds. Based upon electrostatic features of domains that participate in the thiol/disulfide exchange, we prepared mutants of rapeseed Trx-m bearing opposite charges at a single position and subsequently analyzed their action on the activation of rapeseed chloroplast fructose 1,6-phosphate (CFBPase). The replacement of Pro-35 with lysine and glutamic residues enhanced and impaired, respectively, the stimulation of CFBPase relative to the wild-type and the P35A mutant. Furthermore, the shielding of electrostatic interactions with high concentrations of KCl greatly increased and concurrently made indistinguishable the affinity of all variants for CFBPase. The capacity to stimulate the enzyme activity likewise was enhanced concertedly by fructose-1,6-bisphosphate and Ca(2+) but, at variance with the action of KCl, remained sensitive to charges in the side chain of mutants. These results were consistent with a mechanism in which intermolecular electrostatic interactions and intramolecular non-covalent interactions control the formation of the non-covalent complex between reduced Trx and oxidized CFBPase and, in so doing, modulate the thiol/disulfide exchange.
Asunto(s)
Brassica/enzimología , Cloroplastos/enzimología , Fructosa-Bifosfatasa/metabolismo , Tiorredoxinas/química , Tiorredoxinas/metabolismo , Sustitución de Aminoácidos , Brassica/genética , Calcio/metabolismo , Calcio/farmacología , Tiorredoxinas en Cloroplasto , Simulación por Computador , Electroforesis en Gel de Poliacrilamida , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Fructosadifosfatos/metabolismo , Fructosadifosfatos/farmacología , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Cloruro de Potasio/farmacología , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Pliegue de Proteína , Relación Estructura-Actividad , Especificidad por Sustrato/fisiología , Termodinámica , Tiorredoxinas/genéticaRESUMEN
Thioredoxins (Trxs) f and m, as well as their targets chloroplast fructose-1,6-bisphosphatase (FBPase) and NADP+-malate dehydrogenase (NADP-MDH), displayed transcriptional expression in both photosynthetic and non-photosynthetic organs of pea plants (Pisum sativum L. cv. Lincoln) grown for 50 d under normal irradiance. However, whereas Trx m and both target enzymes were poorly expressed in non-photosynthetic tissues, the content of the precursor form of the Trx f-specific mRNA was high in pea roots. In contrast, the translational expression of Trx f was low in this organ. The high FBPase activity in immature seeds, and the low activity of leaves, must be related to high starch synthesis in the first, and with high sucrose formation in the second. The transcriptional expression of FBPase and NADP+-MDH, and to a lesser extent that of Trxs f and m, was inhibited under low irradiance in plants grown under both normal and high temperatures. Pea plants grown at low temperature displayed a high level of mRNAs for Trxs and their targets, especially when the growth was carried out at low light. To a lesser extent, similar behaviour was observed at the protein level. Chloroplasts of mesophyll leaf cells of pea plants grown under saturating light, or under sub-saturating continuous irradiance, showed broken envelopes, distorted structural elements and disorganized starch grains, as a consequence of a photobleaching process and high starch accumulation.