title: Overexpression, purification and enzymatic characterization of a recombinant plastidial glucose-6-phosphate dehydrogenase from barley (Hordeum vulgare cv. Nure) roots.
abstract: In plant cells, the plastidial glucose 6-phosphate dehydrogenase (P2-G6PDH, EC 1.1.1.49) represents one of the most important sources of NADPH. However, previous studies revealed that both native and recombinant purified P2-G6PDHs show a great instability and a rapid loss of catalytic activity. Therefore it has been difficult to describe accurately the catalytic and physico-chemical properties of these isoforms. The plastidial G6PDH encoding sequence from barley roots (Hordeum vulgare cv. Nure), devoid of a long plastidial transit peptide, was expressed as recombinant protein in Escherichia coli, either untagged or with an N-terminal his-tag. After purification from both the soluble fraction and inclusion bodies, we have explored its kinetic parameters, as well as its sensitivity to reduction. The obtained results are consistent with values determined for other P2-G6PDHs previously purified from barley roots and from other land plants. Overall, these data shed light on the catalytic mechanism of plant P2-G6PDH, summarized in a proposed model in which the sequential mechanism is very similar to the mammalian cytosolic G6PDH. This study provides a rational basis to consider the recombinant barley root P2-G6PDH as a good model for further kinetic and structural studies.
authors.affiliation: Dipartimento di Biologia, Università di Napoli "Federico II", Via Cinthia, 80126 Naples, Italy; Université de Lorraine, Unité Mixte de Recherches 1136 Interactions Arbres Microorganismes, F-54500 Vandoeuvre-lès-Nancy, France; INRA, Unité Mixte de Recherches 1136 Interactions Arbres Microorganismes, F-54280 Champenoux, France.
authors.forename: Manuela
authors.initials: M
authors.lastname: Cardi
authors.forename: Kamel
authors.initials: K
authors.lastname: Chibani
authors.forename: Daniela
authors.initials: D
authors.lastname: Castiglia
authors.forename: Donata
authors.initials: D
authors.lastname: Cafasso
authors.forename: Elio
authors.initials: E
authors.lastname: Pizzo
authors.forename: Nicolas
authors.initials: N
authors.lastname: Rouhier
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Jacquot
authors.forename: Sergio
authors.initials: S
authors.lastname: Esposito
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004926:Escherichia coli; D017343:Genes, Plant; D005954:Glucosephosphate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D001467:Hordeum / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D008322:Mammals; D008969:Molecular Sequence Data; D009249:NADP / Q000235:genetics* / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D018517:Plant Roots / Q000378:metabolism*; D018087:Plastids / Q000235:genetics* / Q000378:metabolism; D020033:Protein Isoforms; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D010940:Plant Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D009249:NADP; D005954:Glucosephosphate Dehydrogenase
keywords: Barley roots; G6PDH; OPPP; Plastidial isoform