Diatom plastids possess a phosphoribulokinase with an altered regulation and no oxidative pentose phosphate pathway

Abstract

The chloroplast enzyme phosphoribulokinase (PRK; EC 2.7.1.19) is part of the Calvin cycle (reductive pentose phosphate pathway) responsible for CO(2) fixation in photosynthetic organisms. In green algae and vascular plants, this enzyme is light regulated via reversible reduction by reduced thioredoxin. We have sequenced and characterized the gene of the PRK from the marine diatom Odontella sinensis and found that the enzyme has the conserved cysteine residues necessary for thioredoxin-dependent regulation. Analysis of enzymatic activity of partially purified diatom enzyme and of purified protein obtained by native overexpression in Escherichia coli, however, revealed that under natural redox conditions the diatom enzyme is generally active. Treatment of the enzyme with strong oxidants results in inhibition of the enzyme, which is reversible by subsequent incubation with reducing agents. We determined the redox midpoint potentials of the regulatory cysteine in the PRK from O. sinensis in comparison to the respective spinach (Spinacia oleracea) enzyme and found a more positive redox potential for the diatom PRK, indicating that in vivo this enzyme might not be regulated by thioredoxin. We also demonstrate that in protease-treated diatom plastids, activities of enzymes of the oxidative pentose phosphate pathway are not detectable, thus reducing the need for a tight regulation of the Calvin cycle in diatoms. We discuss our results in the context of rearrangements of the subcellular compartmentation of metabolic pathways due to the peculiar evolution of diatoms by secondary endocytobiosis.

Figure 1.

Alignment of domains of PRKs from different organisms. Odo sin, O. sinensis (accession no. Y08610); Phae tr, P. tricornutum (diatom, EST sequence data at http://avesthagen.sznbowler.com/); Tha ps, T. pseudonana (diatom, genome sequence data at http://genome.jgi-psf.org/thaps1/thaps1.home.html); Vau lit, Vaucheria litoralis (Xanthophyte, AF336986); Galdsu, G. sulphuraria (red alga, AJ012719); Syncyst, Synechocystis PCC6803 (MM77134); Chl rei, C. reinhardtii (M36123); Pis sat, Pisum sativum (Y11248); Ara th, Arabidopsis thaliana (X58149); and Spin ol, spinach (X07654). Numbers above the sequences and at the end of the lines indicate the respective amino acid positions. A, N-terminal region with the regulatory Cys residues. B, C-terminal regions containing additional Cys residues.

Figure 2.

Effects of reducing and oxidizing conditions on the enzymatic activity of the PRK from O. sinensis and spinach stromal extracts. A, Stromal extracts were prepared as described in “Materials and Methods” from plants/algae kept for several hours in the dark or in the light. PRK activity from diatoms generally was not affected by treatment with 50 mm reduced DTT (black columns); in light-treated diatoms, there was a generally higher PRK activity. In a control preparation from spinach plastids, treatment with 50 mm DTT resulted in a strong increase of PRK activity. The bars represent the sd from three independent experiments. B, Inactivation of PRK from O. sinensis stromal extracts by incubation with 5 mm CuCl₂ (final concentration), 125 mm GSSG, 125 mm oxidized DTT. The first column represents the control reactions, the number above the other columns represent the residual activity. C, Reductive reactivation of PRK from O. sinensis stromal extracts that had been inactivated with DTNB. After reduction with reduced DTT, about 90% of the original activity was recovered after 10 min.

Figure 3.

Stoichiometry of the redox reaction of diatom PRK with DTNB. Overexpressed and purified PRK from O. sinensis (5.7 μm) was oxidized stepwise with DTNB, and the amount of released TNB was monitored spectroscopically at λ = 412 nm (white triangles). In parallel, the activity of the PRK was measured in an enzymatic test (black circles). The data clearly show that two molecules TNB are released per molecule DTNB, indicating that DTNB reacts with two Cys in close proximity by forming a disulfide. The reactions were incubated at 15°C to stabilize the diatom enzyme.

Figure 4.

Redox titration of purified recombinant O. sinensis PRK at different pH values. A to E, Oxidized PRK (0.5 μm) was incubated for 75 min at 10°C in oxygen-free buffers (150 mm each) of indicated pH. E_h values were adjusted by different ratios of reduced and oxidized DTT (80 mm total concentration). Enzymatic activity as a measure for reduced enzyme is plotted as a function of the E_h value. Each data point was obtained in two independent experiments. Experimental data were fitted to the Nernst equation for a two-component two-electron redox system. F, E_m versus pH values. From the biphasic redox titrations, two midpoint redox potentials (E_m) were obtained. The lower values (white circles) apparently can be related to the formal standard redox potential of DTT; thus, the higher values (black circles) represent the actual E_m values of the O. sinensis PRK. The broken line represents the E_m values of spinach PRK calculated from E_m and pK_a values of the regulatory cysteins (Hirasawa et al., 1999).

Figure 5.

Enzymatic activities of the OPP enzymes G6PDH (black circles) and 6PGDH (black squares) in isolated plastids from the diatom C. granii. Plastids were pretreated with the protease thermolysin for the time periods indicated on the x axis. After incubation, the protease was inactivated by the addition of excess EGTA compared to the Ca²⁺ concentration and the plastids were broken osmotically. Enzymatic analysis of the stromal extracts demonstrates that the G6PDH and the 6PGDH get degraded by thermolysin treatment, which indicates that the measured activity is due to cytosolic contamination. As a control, the stromal PRK (white circles) is not degraded by thermolysin.