title: SPECIFICITY AND FUNCTION OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE IN A FRESHWATER DIATOM, ASTERIONELLA FORMOSA (BACILLARIOPHYCEAE)(1).
abstract: The plastidic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the only reductive step in the Calvin cycle and exists as different forms of which GapC1 enzyme is present in chromalveolates, such as diatoms. Biochemical studies on diatoms are still fragmentary, and, thus, in this report, GAPDH from the freshwater diatom Asterionella formosa Hassall has been purified and kinetically characterized. It is a homotetrameric enzyme with a molecular mass of ~150 ± 15 kDa. The enzyme showed Michaelis-Menten kinetics with respect to both cofactors, NADPH and NADH, with a 16-fold greater catalytic constant for NADPH. The Km for NADPH was 140 μM, the lowest affinity reported, while the catalytic constant, 815 s(-1) , is the highest reported. The Km for NADH was 93 μM, and the catalytic constant was 50 s(-1) , both are similar to reported values for other types of GAPDH. The GapC1 enzyme, like the Chlamydomonas reinhardtii A4 GAPDH, exhibits a cooperative behavior toward the substrate, 1,3-bisphosphoglyceric acid (BPGA), with both cofactors. Mass spectrometry analysis showed that when GapC1 enzyme was purified without reducing agents, it copurified with a small protein with a mass of 8.2 kDa. This protein was recognized by antibodies against CP12. When associated with this protein, GAPDH displayed a lag that disappeared upon incubation with reducing agent in the presence of either BPGA or NADPH as a consequence of dissociation of the GAPDH/CP12 complex. Thus, as in other species of algae and higher plants, regulation of GapC1 enzyme in A. formosa may occur through association-dissociation processes linked to dark-light transitions.
authors.affiliation: Laboratoire de Bioénergétique et Ingénierie des Protéines, 31 Chemin Joseph Aiguier, 13 402 Marseille Cedex 20 FranceCentre for Ecology and Hydrology, Lancaster Environment Centre, Library Avenue, Bailrigg, Lancaster LA1 4AP, UK.
authors.forename: Jenny
authors.initials: J
authors.lastname: Erales
authors.affiliation: Laboratoire de Bioénergétique et Ingénierie des Protéines, 31 Chemin Joseph Aiguier, 13 402 Marseille Cedex 20 FranceCentre for Ecology and Hydrology, Lancaster Environment Centre, Library Avenue, Bailrigg, Lancaster LA1 4AP, UK.
authors.forename: Brigitte
authors.initials: B
authors.lastname: Gontero
authors.affiliation: Laboratoire de Bioénergétique et Ingénierie des Protéines, 31 Chemin Joseph Aiguier, 13 402 Marseille Cedex 20 FranceCentre for Ecology and Hydrology, Lancaster Environment Centre, Library Avenue, Bailrigg, Lancaster LA1 4AP, UK.
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Maberly
keywords: CP12; GAPDH; GapC1; kinetics