ß CARBOXYLATION ENZYMES IN MARINE PHYTOPLANKTON AND ISOLATION AND PURIFICATION OF PYRUVATE CARBOXYLASE FROMAMPHIDINIUM CARTERAE(DINOPHYCEAE)¹

Abstract

The diatoms, Phaeodactylum tricornutum Bohlin, Cylindrotheca closterium var. californica (Meres.) Reim. & Lewin, Thalassiosira pseudonana Hasle & Heimdal and the prymnesiophyte Pavlova lutheri (Droop), Green, have been shown to contain phosphoenolpyruvate carboxykinase E.C.4.1.1.49. Another diatom Chaetoceros calcitrans (Paulsen) Takano, the chlorophyte Dunaliella tertiolecta Butcher, a rhodophyte Porphyridium cruentum Naegeli and the cyanophyte Anabaena cylindrica Lemmermann, all possessed phosphoenolpyruvate carboxylases E.G.4.1.1.31, which were stimulated by Mn ions and inhibited by malate and aspartate. Two dinoflagellates, Amphidinium carterae Hulbert and Gymnodinium sp., were shown to contain pyruvate carboxylase E.G.6.4.1.1., not previously reported in plants or marine algae. Pyruvate carboxylase was isolated and purified and found to contain biotin and it was inhibited by avidin and could be distinguished from the other two enzymes by complete inhibition by 5 mM Mn ions. The β carboxylating enzymes account for the anaplerotic formation of amino acids and intermediates of the tricarboxylic acid cycle formed during short‐term fixation of CO2.

Size-fractionated phytoplankton carboxylase activities in the Indian sector of the Southern Ocean

“…At the near ice stations, higher PEPCK activity than PEPC or PYRC was observed, especially for the picophytoplankton. Appleby et al hypothesized that PEPCK could be an adaptation to low light intensities because it permits the regeneration of an ATP molecule for each inorganic C atom assimilated (Appleby et al, 1980). This may confirm the hypothesis of low irradiance as a limiting factor at these stations.…”

“…Phytoplankton in marine systems fix inorganic carbon by ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), an enzyme from the Calvin-Benson cycle which uses light as an energy source. The reaction catalysed by Rubisco is not the only carboxylation reaction in autotrophic cells; ␤-carboxylation reactions catalysed by phosphoenolpyruvate carboxylase (PEPC), phosphoenolpyruvate carboxykinase (PEPCK) or pyruvate carboxylase (PYRC) also fix inorganic C using organic components as cosubstrates from the Calvin-Benson cycle or from outside the cell (Appleby et al, 1980;Glover, 1989;Falkowski and Raven, 1997). The interest in ␤-carboxylation reactions is to account for the marked incorporation of inorganic carbon into amino acids and intermediates of the tricarboxylic acid cycle (Mortain-Bertrand, 1988), which is coupled with incorporation of nitrate and ammonium (Yentsch, 1977).…”

“…The interest in ␤-carboxylation reactions is to account for the marked incorporation of inorganic carbon into amino acids and intermediates of the tricarboxylic acid cycle (Mortain-Bertrand, 1988), which is coupled with incorporation of nitrate and ammonium (Yentsch, 1977). Kremer (Kremer, 1981) suggested that ␤-carboxylation reactions are more profitable in terms of energy than the Calvin-Benson (C 3 ) pathway, especially at low-light intensities, because it permits the regeneration of an ATP molecule for each inorganic carbon atom assimilated (Appleby et al 1980). Mortain-Bertrand hypothesized that in Antarctic waters, which are characterized by low light and low temperatures, a high rate of dark fixation by ␤-carboxylation stimulated by high NO 3 or NH 4 may be an important ecological advantage to compensate for low photosynthesis due to the other environmental factors (Mortain-Bertrand, 1988).…”

Inorganic carbon concentrating mechanisms in free‐living and symbiotic dinoflagellates and chromerids

“…Ten percent of the organic label is in the C 4 dicarboxylate aspartate, which is produced by carboxylation by a C 3 + C 1 (anaplerotic) carboxylase. This enzyme in basal, peridinin-containing, dinoflagellates can be pyruvate carboxylase in Amphidinium carterae, Amphidinium operculatum, and Scripsiella trochoidea (Appleby et al 1980, Descolas-Gros and Fontugne 1985, Descolas-Gros and Oriol 1992, phosphoenolpyruvate carboxylase in Prorocentrum micans (Descolas-Gros and Oriol 1992), and phosphoenolpyruvate carboxykinase in the non-photosynthetic Cryptothecodinium cohnii (Descolas-Gros and Oriol 1992). The 5 s 14 C-inorganic C labeling does not yield products associated with RuBisCO oxygenase activity (Streamer et al 1993); this is consistent with the O 2 -affinity characteristics of light stimulated 18 O 2 uptake that resembles a water-water cycle (e.g., Mehler-Peroxidase or flavodi-iron O 2 uptake) rather than RuBisCO oxygenase (Leggat et al 1999, Badger et al 2000.…”

Photoadaptation in marine phytoplankton: variations in ribulose 1,5-bisphos-phate activity

“…The genetics, structure and regulation of RuBPCase has been studied extensively in higher plants (e.g. Lorimer 1981, Yoeh et al 1981, Miziorko & Lorimer 1983, Akazawa et al 1984, Kobza & Seemann 1988, but not in microalgae (Estep et al 1978, Appleby et al 1980, Hobson et al 1985, Plumley et al 1986, Newman & Cattolico 1987. Despite the obvious biochemical and physiological importance of RuBPCase, there have been surprisingly few systematic studies on the relationships between RuBPCase, or the 8-carboxylating Li et al 1984, Smith & Platt 1985, Mortain-Bertrand e~ al.…”