title: Expression and characterization of a cytosolic glucose 6 phosphate dehydrogenase isoform from barley (Hordeum vulgare) roots.
abstract: In plant cells, glucose 6 phosphate dehydrogenase (G6PDH-EC 1.1.1.49) regulates the oxidative pentose phosphate pathway (OPPP), a metabolic route involved in the production of NADPH for various biosynthetic processes and stress response. In this study, we report the overexpression of a cytosolic G6PDH isoform from barley (Hordeum vulgare) roots in bacteria, and the biochemical characterization of the purified recombinant enzyme (HvCy-G6PDH). A full-length cDNA coding for a cytosolic isoform of G6PDH was isolated, and the sequence was cloned into pET3d vector; the protein was overexpressed in Escherichia coli BL21 (DE3) and purified by anion exchange and affinity chromatography. The kinetic properties were calculated: the recombinant HvCy-G6PDH showed KMs and KINADPH comparable to those observed for the enzyme purified from barley roots; moreover, the analysis of NADPH inhibition suggested a competitive mechanism. Therefore, this enzyme could be utilised for the structural and regulatory characterization of this isoform in higher plants.
authors.affiliation: Università di Napoli "Federico II", Dipartimento di Biologia, Via Cinthia, 6, I-80126 Napoli, Italy; CNR-IBBR (Istituto di Bioscienze e BioRisorse), Via Università, 133, I-80055 Portici (NA), Italy(1).
authors.forename: Daniela
authors.initials: D
authors.lastname: Castiglia
authors.affiliation: Università di Napoli "Federico II", Dipartimento di Biologia, Via Cinthia, 6, I-80126 Napoli, Italy.
authors.forename: Manuela
authors.initials: M
authors.lastname: Cardi
authors.affiliation: CNR-IBBR (Istituto di Bioscienze e BioRisorse), Via Università, 133, I-80055 Portici (NA), Italy(1).
authors.forename: Simone
authors.initials: S
authors.lastname: Landi
authors.affiliation: Università di Napoli "Federico II", Dipartimento di Biologia, Via Cinthia, 6, I-80126 Napoli, Italy.
authors.forename: Donata
authors.initials: D
authors.lastname: Cafasso
authors.affiliation: Università di Napoli "Federico II", Dipartimento di Biologia, Via Cinthia, 6, I-80126 Napoli, Italy. Electronic address: sergio.esposito@unina.it.
authors.forename: Sergio
authors.initials: S
authors.lastname: Esposito
mesh_terms: D000595:Amino Acid Sequence; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001467:Hordeum / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism*; D010802:Phylogeny; D018517:Plant Roots / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D011994:Recombinant Proteins; D009249:NADP; D005954:Glucosephosphate Dehydrogenase
keywords: Anion exchange chromatography; Barley; Competitive inhibition; G6PDH; NADPH; Pentose phosphate pathway; pET3d