Carbon dioxide assimilation in cyanobacteria: regulation of ribulose, 1,5-bisphosphate carboxylase

Abstract

Cyanobacteria assimilate carbon dioxide through the Calvin cycle and therefore must regulate the activity of ribulose 1,5-bisophosphate carboxylase. Using an in situ assay, as well as measuring the activity in crude, partially purified, and homogeneous preparations, we can show that a number of phosphorylated intermediates exert a regulatory role. Three diverse organisms, Agmenellum quadruplicatum, Aphanocapsa 6714, and Anabaena sp. CA, were studied, and it was found that the in situ and cell-free carboxylase activities were particularly affected by low levels ofphosphogluconate and reduced nicotinamide adenine dinucleotide phosphate. There was a marked activation by these ligands when the inactive enzyme was assayed in the presence of low levels of bicarbonate, a result significantly different from a previous report. Moreover, the fully activated enzyme was inhibited by phosphogluconate. In situ Anabaena CA carboxylase activity exhibited a particular capacity for activation by phosphogluconate and reduced nicotinamide adenine dinucleotide phosphate. However, activation of the crude, partially purified, or homogeneous Anabaena CA carboxylase by phosphogluconate and reduced nicotinamide adenine dinucleotide phosphate was significantly decreased when compared with enzyme activity in permeabilized cells. It appears that the microenvironment or the conformation of the enzyme within the cell may be significantly different from that of the isolated enzyme.Cyanobacteria (blue-green algae) form organic matter by assimilating carbon dioxide. The enzyme which catalyzes this primary CO2 assimilation is ribulose 1,5-bisphosphate (Rbu-P2) carboxylase (3-phospho-D-glycerate carboxylase [dimerizing]; EC 4.1.1.39). Various phosphorylated intermediates, particularly those characteristic of photosynthetic metabolism, have been shown to either inhibit or activate the carboxylase. Inhibition by 6-phosphogluconate, NADPH, or fructose bisphosphate is pronounced when the enzyme is activated by preincubation at high levels of bicarbonate (20 to 50 mM) in the presence of magnesium (3,7,21). In contrast, upon incubation at low levels of bicarbonate (about 1 mM), the carboxylase is relatively inactive, and phosphorylated metabolites (particularly NADPH and phosphogluconate) markedly activate the inactive enzyme if included in the preincubation mixture (4-6, 8, 9, 15, 18). Such behavior yielded a plausible hypothesis for the physiological regulation of this key enzyme based upon the known concentrations and fluctuations of these metabolites within the chloroplast (1, 14).The cyanobacteria are procaryotic organisms that resemble the chloroplasts of photosynthetic eucaryotes in that they possess the capacity to split water and thus evolve oxygen via photosystem II-directed photosynthesis. Moreover, kinetic studies of "CO2 fixation and 32P incorporation into Aphanocapsa strains 6308 and 6714 (16, 17) showed a rapid increase in the concentration of phosphogluconate and changes in the concentrations of sugar mono-and diphosphates when cells wer...

The Carboxysomes (Polyhedral Bodies) of Autotrophic Prokarygtes

“…T h e immediate cessation of cyanobacterial CO, fixation upon transfer from light to dark is accompanied by the appearance of 6PGlu among rapid 14C-and 32P-labelling products ( (Stanier & Cohen-Bazire, 1977). Tabita & Colletti (1979) have found inhibition of activated RuBisCO by exogenous 6PGlu, FBP, fructose 6-phosphate, NADPH and ATP, and activation of inactive RuBisCO by thesecompounds when tested in toluene-permeabilized whole cells of three cyanobacteria. T h e value of using permeabilized whole cells to study RuBisCO regulation in situ has been confirmed with Rhodospirillum rubrum : previous observations in vitro were generally found with the permeabilized cells (Storro & McFadden, 1983).…”

“…14C-CABP-labelling of both cytoplasmic and carboxysomal RuBisCO was found after fractionation of the permeabilized cells, indicating that carboxysomal RuBisCO is capable of being activated in situ (Cannon, 1982). T h e action of toluene and chloroform (Tabita, Caruso & Whitman, I 978;Tabita & Colletti, 1979;Cannon, 1982) in rendering cell walls and membranes permeable to RuBP and CABP is evident. However, the effects of these agents on carboxysome membranes, which may lack lipids (Biedermann & Westphal, 1979), is unknown.…”

“…CO, fixation by autotrophic prokaryotes may also be influenced by such metabolites on RuBisCO. Inhibition of CO, fixation by 6PGlu occurs with purified, high HCO; (20 mM)-activated 8L8S RuBisCOs from cyanobacteria (Tabita & McFadden, I 9-72 ;Codd & Stewart, 1977 b ;Tabita & Colletti, 1979), Pseudomonas oxalaticus (Lawlis, Gordon & McFadden, 1979), Thiobacillus neapolitanus (Snead & Shively, 1978) and photosynthetic bacteria (McFadden & Purohit, 1978;Gibson & Tabita, I977a, 6). T h e only exceptions to these findings to date have been the 2L RuBisCO from Rhodospirillum rubrum (Tabita & McFadden, 1972;McFadden & Purohit, 1978) and the form I1 (6L) enzymes from Rhodopseudomonas sphaeroides (Gibson & Tabita, 1977 a ) and Rhodopseudomonas capsulata (Gibson & Tabita, 19776).…”

“…When incubated in the inactive form with low HCO; ( I mM), the 8L8S enzymes from the cyanobacteria Agmenellum quadruplicatum, Anabaena CA and Aphanocapsa 6714, and the zL R . rubrum enzyme, are activated by 6PGlu and NADPH (Tabita & Colletti, 1979;Tabita, 1981). In this respect, these bacterial enzymes resemble plant RuBisCO (Chu & Bassham, 1973.…”

Molecular and cellular regulation of autotrophic carbon dioxide fixation in microorganisms.

“…This, combined with an unusual asymmetric appearance of the enzyme in the electron microscope, has led to low-molecular-weight estimates of the cyanobacterial enzymes since sedimentation equilibrium analysis of the Synechococcus enzyme yields a molecular weight of 530,000. The enzyme from cyanobacteria contains a typical L8S8 structure (11,65,323,337). However, prior studies had indicated that the Agmenellum and Anabaena enzymes might be composed of only large subunits (L8 structure) (333, 334) and the enzyme from the extreme halophile Aphanothece halophytica was reported to be a tetramer (L4) (60).…”