Plastidic P2 glucose-6P dehydrogenase from poplar is modulated by thioredoxin m-type | Plastidic P2 glucose-6P dehydrogenase from poplar is modulated by thioredoxin m-type: Distinct roles of cysteine residues in redox regulation and NADPH inhibition
2016
Cardi, Manuela | Zaffagnini, Mirko | de Lillo, Alessia | Castiglia, Daniela | Chibani, Kamel | Gualberto, José Manuel | Rouhier, Nicolas | Jacquot, Jean-Pierre | Esposito, Sergio | Dipartimento Biol ; University of Naples Federico II = Università degli studi di Napoli Federico II | Dipartimento Farm & Biotecnol ; Alma Mater Studiorum Università di Bologna = University of Bologna (UNIBO) | Interactions Arbres-Microorganismes (IAM) ; Institut National de la Recherche Agronomique (INRA)-Université de Lorraine (UL) | Central European Institute of Technology [Brno] (CEITEC MU) ; Brno University of Technology [Brno] (BUT) | Université de Strasbourg (UNISTRA) | Legge Regionale della Campania CUP E69D15000270002, 5/2002 ; French Ministry of Foreign Affairs 672700K ; Project FORGIARE (Formazione Giovani alla Ricerca) by Compagnia di San Paolo V-10/FORG/ST/2012/5 ; French National Research Agency (ANR) as part of the "Investissements d'Avenir" program (Lab of Excellence ARBRE) UMR1136 ANR-11-LABX-0002-01 | ANR-11-LABX-0002,ARBRE,Recherches Avancées sur l'Arbre et les Ecosytèmes Forestiers(2011) | ANR-11-LABX-0057,MitoCross,Expression et coordination des génomes dans les fonctionnements et dysfonction-nements mitochondriau(2011)
A cDNA coding for a plastidic P2-type G6PDH isoform from poplar (Populus tremula x tremuloides) has been used to express and purify to homogeneity the mature recombinant protein with a N-terminus His-tag. The study of the kinetic properties of the recombinant enzyme showed an in vitro redox sensing modulation exerted by reduced DTT. The interaction with thioredoxins (TRX5) was then investigated.Five cysteine to serine variants (C145S - C175S - C183S - C195S - C242S) and a variant with a double substitution for Cys(175) and Cys(183) (C175S/C183S) have been generated, purified and biochemically characterized in order to investigate the specific role(s) of cysteines in terms of redox regulation and NADPH - dependent inhibition.Three cysteine residues (C-145, C-194, C-242) are suggested to have a role in controlling the NADI)* access to the active site, and in stabilizing the NADPH regulatory binding site.Our results also indicate that the regulatory disulfide involves residues Cys(175) and Cys(183) in a position similar to those of chloroplastic P1-G6PDHs, but the modulation is exerted primarily by TRX m-type, in contrast to P1-G6PDH, which is regulated by TRX f.This unexpected specificity indicates differences in the mechanism of regulation, and redox sensing of plastidic P2-G6PDH compared to chloroplastic Pl - G6PDH in higher plants. (C) 2016 Elsevier Ireland Ltd. All rights reserved.
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