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First published online February 25, 2005; 10.1104/pp.104.055285 Plant Physiology 137:911-920 (2005) © 2005 American Society of Plant Biologists Diatom Plastids Possess a Phosphoribulokinase with an Altered Regulation and No Oxidative Pentose Phosphate Pathway1Institute of Plant Biochemistry, University of Düsseldorf, 40225 Duesseldorf, Germany (A.K.M.); Plant Physiology, Department of Biology/Chemistry, University of Osnabrueck, 49069 Osnabrueck, Germany (N.W.); and Department of Biology, Postfach M611, University of Konstanz, 78457 Konstanz, Germany (P.G.K.)
The chloroplast enzyme phosphoribulokinase (PRK; EC 2.7.1.19) is part of the Calvin cycle (reductive pentose phosphate pathway) responsible for CO2 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.
The enzyme phosphoribulokinase (PRK; EC 2.7.1.19) is found in most phototrophic organisms. Next to Rubisco, it is one of the key enzymes of the reductive pentose phosphate pathway as it catalyzes the only reaction by which Calvin cycle intermediates can be provided for further CO2 fixation. The kinase catalyzes the in-line transfer of the  -phosphoryl group from ATP to the C-1 hydroxyl of Ru-bP, thereby forming Ru-1,5-bP, the acceptor molecule for CO2. Class I and II PRK enzymes are known (Martin and Schnarrenberger, 1997 ). Class I enzymes are found in protobacteria and in some cyanobacteria (Badger et al., 2002), while Class II PRK is found in cyanobacteria, in eukaryotic algae, and in land plants. The PRK of the latter group typically is a dimer of identical 40-kD subunits, which is located in the stroma of the chloroplasts (Porter et al., 1986). In plants, PRK activity is linked to the light-driven electron transport at the thylakoid membrane via the ferredoxin/thioredoxin system (Wolosiuk and Buchanan, 1978; Buchanan, 1980; Porter et al., 1988; Jacquot et al., 1997). During illumination, plastidic thioredoxins are constantly reduced by the ferredoxin-thioredoxin oxidoreductase. The reduced form of thioredoxin is then able to reduce an intramolecular regulatory disulfide of the oxidized, inactive PRK, resulting in an immediate activation of the kinase (Hirasawa et al., 1999). Higher plant PRKs possess four Cys; the regulatory disulfide of PRK is formed by the two N-terminal Cys (Cys-16 and Cys-55 in spinach [Spinacia oleracea]; Porter et al., 1988; Porter and Hartman, 1990). Several plastidic enzymes in plants are light regulated via thioredoxin (for review, see Ruelland and Miginiac-Maslow, 1999), but it is an exceptional feature of the PRK that both regulatory Cys are located in the active site. Apparently, Cys-55 has a modest facilitative role in catalysis (Porter et al., 1988; Porter and Hartman, 1990). A systematic investigation on the oxidation-reduction midpoint potentials (Em) of the disulfide/dithiol couple of enzymes regulated by thioredoxin revealed that PRK possesses the most positive potential (–290 mV for spinach PRK; Hirasawa et al., 1998, 1999) and therefore is easier to activate than the other light-regulated enzymes of the Calvin cycle. It has been demonstrated that in the green alga Chlamydomonas reinhardtii the PRK can form regulatory complexes with the enzyme glyceraldehyde phosphate dehydrogenase (GAPDH) and a small protein named CP12 (Wedel et al., 1997; Mouche et al., 2002; Lebreton et al., 2003). Recently it has been shown that the PRK from the cyanobacterium Synechococcus sp. PCC 7042 is permanently active but can be inactived by strong oxidants (Kobayashi et al., 2003).
A tight regulation of Calvin cycle enzymes via the ferredoxin/thioredoxin system is essential, as this pathway constitutes the interface between the light reactions at the thylakoid membranes and the utilization of the produced metabolites for other biosynthetic purposes. For example, the oxidative pentose phosphate pathway (OPP) in land plant plastids (Schnarrenberger et al., 1973
Diatoms (Bacillariophyceae) contribute significantly to the total O2 evolution and primary biomass production in oceans and other aquatic ecosystems (Falkowski et al., 1998
With the exception of the plastid-encoded genes for the large and small subunits of the Rubisco and the GAPDH (Hwang and Tabita, 1991
In this study, we investigated the redox state of the PRK in diatom plastids under different redox conditions. We determined the redox properties of the heterologously expressed and purified PRK from the diatom O. sinensis. Using the redox poising technique (Hutchison and Ort, 1995
Features of the PRK from O. sinensis
We cloned and sequenced the complete gene encoding the PRK of the diatom O. sinensis from a cDNA library using degenerate primers, which were derived from homologous Prk regions of other organisms. To verify this sequence, we amplified the identical genomic DNA fragment from nuclear DNA of O. sinensis, showing that there is no intron in the entire nuclear Prk gene in O. sinensis. Comparison of the derived amino acid sequence with PRK sequences from land plants, cyanobacteria, red algae, and other diatoms revealed a sequence similarity of 60% to 80% identical amino acids. As in most other plastid PRKs, the O. sinensis enzyme contains four Cys residues present in the primary sequence (Fig. 1; positions 19, 63, 224, and 236). The first two residues, located in the N-terminal part of the polypeptide, are in conserved positions and are found in all sequences analyzed so far. It has been shown that the first two Cys are responsible and indispensable for the redox regulation in land plant plastids (Porter et al., 1988
The PRK protein from O. sinensis contains a bipartite presequence (data not shown), which is structurally very similar to other plastid-targeting presequences in diatoms (Kroth, 2002 ). Typical for such presequences is an N-terminal signal peptide with a basic amino acid residue and a hydrophobic domain, followed by a domain showing features of transit peptides of land plant plastid preproteins (Kilian and Kroth, 2005). By gel filtration experiments, we found that the PRKs from different diatoms (O. sinensis, Coscinodiscus granii, and P. tricornutum) are homodimers with an apparent molecular mass of 44 kD/subunits on SDS gels (data not shown). This is identical to PRK enzymes from land plants but differs from previously published data for the PRK of the haptophyte Heterosigma carterae (Hariharan et al., 1998).
In previous works, it has been shown that the enzymatic activity of PRK from land plants is strongly dependent on the redox state and that a full activation of PRK in vivo is found only in illuminated plants (Wedel and Soll, 1998
As the diatom PRK possesses Cys residues located in the same position as the regulatory Cys in land plant PRKs, we checked whether the enzymatic PRK activity in O. sinensis was in any way influenced by oxidation or by reduction. We isolated crude stromal extracts from previously illuminated diatoms and treated them with oxidizing agents like copper in the presence of oxygen, oxidized glutathione (GSSG), or oxidized DTT and found a decrease of PRK activity to different degrees (Fig. 2B). The degree of inhibition corresponds to the redox potential of the oxidizing agents (DTT –327 mV; Lees and Whitesides, 1993 ; GSSG –250 mV; Lundström and Holmgren, 1983). Furthermore, PRK activity was completely abolished by treatment of the stromal extract with the strong oxidant dithiobisnitro-benzoic acid (DTNB, Ellman's reagent; Brandes et al., 1996). Enzymatic activity of oxidized inactivated PRK could be restored by treatment with reducing agents (Fig. 2C), thus indicating that PRK inhibition was not due to unspecific oxidation and inactivation of the enzyme.
To exclude unknown interfering factors or proteins within the stromal extract and to investigate the redox regulation of PRK in detail, we fused the cDNA encoding the mature part of the PRK protein from O. sinensis to a gene coding for an intein tag (intein plus a chitin-binding domain) for affinity chromatography. The putative N terminus of the mature O. sinensis PRK protein (position 43) was inferred by comparison of the complete amino acid sequence of the PRK with (1) other mature PRK sequences (Milanez and Mural, 1988
Analysis of Diatom PRK Inactivation by DTNB
Disulphide bridge formation requires a close proximity of both sulfhydryl groups involved. Such proximity has been successfully demonstrated for PRK from spinach utilizing the dithiol DTNB (Porter et al., 1988
Determination of the Midpoint Redox Potential of the PRK from Diatoms
As the PRK from O. sinensis apparently can be modulated reversibly by oxidation, we need to question why the enzyme is permanently active in stromal extracts. Different secondary or tertiary structure might be responsible for conformational changes that results in a different redox potential of the diatom enzyme compared to land plant enzymes. Therefore, we determined the midpoint redox potential of the PRKs from spinach and O. sinensis by redox titration (redox poising; Hutchison and Ort, 1995 Optimization of the protocol for the recombinant diatom PRK revealed that the enzymatic activity was not stable when the enzyme was incubated at 25°C; therefore, we incubated at 10°C with various DTTox/red ratios. Under anaerobic conditions, we observed stable PRK activity for 2 h or more. This was sufficient to achieve redox equilibrium between DTT and the enzyme, a process that takes up to 30 min (this is about 2 times faster than observed for the spinach enyzme). We have determined the Em values for the PRK at various pH values (Fig. 4). The maximum activity obtained at the lowest Eh values was identical to the maximum activity of the completely reduced purified enzyme. In contrast to the results of the spinach enzyme (not shown), we found that the curves did not fit exactly to a Nernst equation for a two-electron single-component redox reaction. Especially at pH 7.5 and 8.0, we obtained a biphasic curve resulting in the calculation of 2 putative Em values. Plotting the Em values of the spinach and the diatom PRK as a function of the pH shows that the obtained Em of the diatom PRK are either above or below the value of spinach PRK. Two Em values indicate that either additional Cys within the PRK might form disulphide bridges or that one of the Em values represents the formation of artificial mixed dimers of enzyme and DTT. Therefore, we also plotted the respective values of DTT, which were very similar to the more negative Em values of the diatom PRK. This indicates that the higher value of –257 mV, pH 7.0, in fact represents the actual Em value of the diatom PRK at pH 7.
There Is Apparently No OPP in Diatom Plastids
To analyze the redox regulation properties of enzymes of the OPP, we measured the activity of 2 key enzymes, the G6PDH and the 6-phosphogluconate dehydrogenase (6PGDH). We purified intact plastids from the diatoms O. sinensis and C. granii and found low respective activities in the stromal extracts. Both enzymes apparently were not affected by changing redox conditions (data not shown). This result indicated that we might have measured the cytosolic isoenzymes attached to the outside of the envelope membranes of the plastids; therefore, we treated the plastids with the protease thermolysin (Cline et al., 1984
We found the PRK from O. sinensis to be active in diatom plastids independent of whether the algae were illuminated prior to isolation or whether they were kept in the dark (Fig. 2). This was surprising as the respective enzyme from land plants is usually inactive and gets activated in the light via reduction by reduced thioredoxin (Anderson, 1973 ; Gardemann et al., 1983). From the nucleotide sequence, it was obvious that the respective Cys residues involved in redox regulation of the land plant enzymes (Porter et al., 1988) are present in the diatom PRK. The distance and the amino acid sequence between the regulatory Cys are strongly conserved within land plants and green algae (see also Fig. 1). For the PRK of O. sinensis, however, we found an insertion of 5 amino acids between Cys 19 and 63. The PRK sequences of the rhodophyte Galdieria sulphuraria and of some cyanobacteria show deletions between the regulatory Cys. Interestingly, the enzymes from cyanobacteria (Kobayashi et al., 2003) and from G. sulphuraria are also possibly not redox regulated (R. Scheibe, personal communication). In contrast to thioredoxin, where the redox active Cys are already in proximity at the level of the primary enzyme structure, the orientation of regulatory Cys of PRK is dependent on the protein fold. Therefore, it is possible that the insertion in the diatom enzyme affects the distance between the regulatory Cys. Enzymatic analyses support this view: We were able to show reversible inactivation of the enzyme by strong oxidants and to recover activity by subsequent reduction of the enzyme. Oxygen, which is believed to inactivate the land plant PRK, apparently is not strong enough to form a disulfide bridge, thus leaving the enzyme in a permanently active mode. There are reports that algae and cyanobacteria accumulate higher concentrations of H2O2 (Badger et al., 2000); according to our data, this is probably not true for diatom plastids. A likely reason for the lost ability of the diatom PRK to be regulated by thioredoxin could be the apparently missing OPP in diatom plastids. In land plants, both reductive and oxidative pentose phosphate pathways have to be controlled strictly to prevent an energy-consuming futile cycle. Since the oxidative pathway is missing in diatom plastid, such a stringent control appears to be no longer necessary.
Previous work on light regulation of diatom enzymes indicated that thioredoxin might not play a role in diatom plastids. Several enzymes in diatoms, like the chloroplast ATPase (Pancic and Strotmann, 1993
Another question is how diatoms survive without plastidic oxidative pentose pathway. In land plants, the OPP is closely connected to the biosynthesis of fatty acids (Jensen, 1985
Diatoms and other algae-like euglenophytes (brown algae, etc.) are supposed to have evolved by so-called secondary endocytobiosis. Substantial evidence suggests that all plastids can be traced back to a single primary endocytobiosis, which led to the evolution of plastids in green algae, land plants, red algae, and glaucophytes; (see Delwiche and Palmer, 1997
Materials
If not stated otherwise, all chemicals used were p.a. grade. Odontella sinensis was cultured according to Pancic et al. (1990)
The gene encoding the PRK was cloned from a cDNA library of the diatom O. sinensis (Pancic and Strotmann, 1993
PRK activity of stromal extracts or purified protein was measured according to Racker (1957)
Stromal extracts were obtained from purified plastids by pelleting the plastids at 1,000g for 3 min and resuspension in 50 mM Bicine-KOH, pH 8.0. After incubation for 15 min on ice, the thylakoid membranes were removed by centrifugation at 20,000g for 15 min. Stromal fractions equivalent to 150 to 250 µg chlorophyll were incubated for 10 min at room temperature with 50 mM reduced DTT plus 10 mM MgCl2, 5 mM CuCl2, 125 mM GSSG, and 125 mM oxidized DTT.
The PRK gene was cloned into the vector pTYB1 (New England Biolabs, Beverly, MA). We amplified the region encoding the mature part of the PRK by PCR creating new NdeI (5') and SapI (3') restriction sites and ligated this fragment into the vector resulting in an in-frame fusion to a reading frame encoding an intein tag and a chitin-binding domain. This vector PTYmatPRK was transformed into E. coli strain BL21 (DE3) codonplus (Stratagene, La Jolla, CA). The bacteria were grown in Luria-Bertani medium including 0.2 Ara and 10% (v/v) glycerol at 30°C up to an optical density of 0.7, and then cooled to 16°C. After addition of isopropylthio- Sixty grams of cells were suspended in buffer (20 mM HEPES/KOH, pH 7.85; 1 mM NaCl, 1 mM EDTA, 6 mm MgCl2, and 1 mM phenlymethane sulfonfluorid) in a glass/teflon homogenizer. After addition of Dnase I, the cells were ruptured in a French press (Aminco International, Lake Forrest, CA) with a constant pressure of 1,200 psi. Cell debris was removed by centrifugation (15 min, 6,000g, 4°C). The supernatant was again centrifuged (30 min, 12,000g, 4°C) and the supernatant used for further purification by affinity chromatography on chitin agarose. After addition of 0.1% (v/v) Triton X-100, the extract was filtered through a 0.2-µm nylon membrane and loaded onto a column (2.2 x 5 cm) containing 20 mL chitin agarose beads. The flow through fraction was checked by western blots to ensure proper binding of the fusion protein. The column was washed with 300 mL washing buffer A (20 mM HEPES/KOH, pH 7.85, 1 M NaCl, 1 mM EDTA, 0.1% Triton X-100), followed by washing with 60 mL washing buffer B (20 mM HEPES/KOH, pH 7.5, 50 mM NaCl, 1 mM EDTA). The column was then washed with 60 mL of the same buffer containing 60 mM DTT, sealed, and stored at 4°C for 16 to 20 h. During this, the reducing conditions resulted in a protein splicing releasing the mature part of the PRK while the intein tag remains bound to the column. Elution of the PRK by 20 mM HEPES, pH 7.5, 50 mM NaCl; 1 mM EDTA resulted in a sharp peak fraction containing PRK protein with a purity of approx 95%. Further purification was achieved by ion-exchange chromatography on a prepacked Resource Q column (2 mL, Amersham, Buckinghamshire, UK) equilibrated in 20 mM BisTris propane, pH 7.5, 1 mM EDTA, and 50 mM NaCl. After a washing step to decrease unspecific binding, the PRK was eluted in a linear NaCl gradient (50–1,000). The eluate was concentrated by high pressure filtration, dialyzed 4 times against storage buffer (50 mM Bicine/KOH, pH 8.0, and 20% [v/v] glycerol) and stored at –80°C.
Chloroplasts from 2 kg of spinach were prepared as described above. The plastids were then broken by addition of 300 to 400 mL of 50 mM Bicine/KOH, pH 8.0, 2.5 mM EDTA, 1 mM phenlymethane sulfonfluorid, 5 mM DTT, 5 mM MgCl2, and 10 mM NaCl for 10 min on ice. The suspension was then centrifuged for 20 min at 12,000g. The second pellet obtained from a fractionated ammonium sulfate precipitation (35% and 65%) was resuspended in 50 mM Bicine/KOH, pH 8.0, 1 mM EDTA, and 10 mM DTT (BED) buffer and diluted to a maximum current of 5 mS/cm. Further purification of the PRK was performed according to Brandes et al. (1996)
Spinach thioredoxins f and m were expressed from plasmids Trxf and Trxm (Stumpp et al., 1999
The concentration of purified PRK and thioredoxin was determined by the Bradford Assay (Bio-Rad Laboratories, Hercules, CA). Substrates were measured directly in a photometer: ATP (
PRK from Odontella was inhibited by DTNB according to Porter et al. (1988)
Solutions of defined pre-equilibrated redox potentials (Eh) were generated in oxygen-free buffer according to Hutchison and Ort (1995) Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession number Y08610.
The authors thank Prof. Dr. Heinrich Strotmann for support and helpful discussions, Prof. Dr. David Knaff for helpful suggestions, Prof. Dr. Toru Hisabori and Dr. Johann Lavaud for critical comments on the manuscript, and Renate Thelen and Katharina Weyrauch for help in cloning the Prk gene. Received October 19, 2004; returned for revision December 1, 2004; accepted December 4, 2004.
1 This work was supported by the University of Konstanz, the Deutsche Forschungsgemeinschaft (Transregio-SFB TR1, project Kr 1661–2, grant to P.G.K.), and by the European community (MARGENES, grant no. QLRT–2001–01226 to P.G.K.). 
2 Present address: Henkel KGaA, Division of Enzyme Technology, Henkelstrasse 67, 40191 Düsseldorf, Germany.
3 Present address: Hofkamp 7, 24145 Kiel, Germany. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.055285. * Corresponding author; e-mail peter.kroth{at}uni-konstanz.de; fax 49–7531–883042.
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TOP
ABSTRACT
RESULTS
= 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.
of DTT; thus, the higher values (black circles) represent the actual Em values of the O. sinensis PRK. The broken line represents the Em values of spinach PRK calculated from Em and pKa values of the regulatory cysteins (Hirasawa et al., 1999
-glucan chrysolaminarin in cytosolic vacuoles instead), sugar phosphates have to be imported into their plastids in the dark as well, making an OPP more or less useless. Reduction equivalents might easily be imported into the plastids by shuttle systems known from land plant plastids (Eicks et al., 2002
= 15.4 mmol–1 cm–1), TNB2– (412 nm, 
