title: Plastidic P2 glucose-6P dehydrogenase from poplar is modulated by thioredoxin m-type: Distinct roles of cysteine residues in redox regulation and NADPH inhibition.
abstract: 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 (TRXs) was then investigated. Five cysteine to serine variants (C145S - C175S - C183S - C195S - C242S) and a variant with a double substitution for Cys175and Cys183(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 (C145, C194, C242) are suggested to have a role in controlling the NADP+access to the active site, and in stabilizing the NADPH regulatory binding site. Our results also indicate that the regulatory disulfide involves residues Cys175and Cys183in 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 P1-G6PDH in higher plants.
authors.affiliation: Dipartimento di Biologia, Univ. di Napoli "Federico II", I-80126 Napoli, Italy.
authors.forename: Manuela
authors.initials: M
authors.lastname: Cardi
authors.affiliation: Dipartimento di Farmacia e Biotecnologie, Univ. di Bologna, I-40126 Bologna, Italy.
authors.forename: Mirko
authors.initials: M
authors.lastname: Zaffagnini
authors.affiliation: Dipartimento di Biologia, Univ. di Napoli "Federico II", I-80126 Napoli, Italy.
authors.forename: Alessia
authors.initials: A
authors.lastname: De Lillo
authors.affiliation: Dipartimento di Biologia, Univ. di Napoli "Federico II", I-80126 Napoli, Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: Castiglia
authors.affiliation: Université de Lorraine, UMR 1136 Interactions Arbres/Microorganismes, Faculté des Sciences et Technologies, 54506, Vandoeuvre-lès-Nancy, France; INRA, UMR 1136 Interactions Arbres/Microorganismes, Centre INRA Nancy Lorraine, 54280, Champenoux, France.
authors.forename: Kamel
authors.initials: K
authors.lastname: Chibani
authors.affiliation: Université de Strasbourg, Institut de Biologie Moléculaire Des Plantes (IBMP), CNRS-UPR 2357, 67084 Strasbourg, France.
authors.forename: José Manuel
authors.initials: JM
authors.lastname: Gualberto
authors.affiliation: Université de Lorraine, UMR 1136 Interactions Arbres/Microorganismes, Faculté des Sciences et Technologies, 54506, Vandoeuvre-lès-Nancy, France; INRA, UMR 1136 Interactions Arbres/Microorganismes, Centre INRA Nancy Lorraine, 54280, Champenoux, France.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Rouhier
authors.affiliation: Université de Lorraine, UMR 1136 Interactions Arbres/Microorganismes, Faculté des Sciences et Technologies, 54506, Vandoeuvre-lès-Nancy, France; INRA, UMR 1136 Interactions Arbres/Microorganismes, Centre INRA Nancy Lorraine, 54280, Champenoux, France.
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Jacquot
authors.affiliation: Dipartimento di Biologia, Univ. di Napoli "Federico II", I-80126 Napoli, Italy. Electronic address: sergio.esposito@unina.it.
authors.forename: Sergio
authors.initials: S
authors.lastname: Esposito
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry / Q000502:physiology; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000037:antagonists & inhibitors / Q000737:chemistry; D010084:Oxidation-Reduction; D010427:Pentose Phosphate Pathway; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D018087:Plastids / Q000378:metabolism*; D032107:Populus / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D013879:Thioredoxins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*
chemical_list: D010940:Plant Proteins; D011994:Recombinant Proteins; D013879:Thioredoxins; D009249:NADP; D005954:Glucosephosphate Dehydrogenase; D003545:Cysteine
keywords: Cysteine; Oxidative pentose phosphate pathway; Populus; Thioredoxins