an important but neglected Calvin cycle enzyme

Journal of Experimental Botany, Vol. 50, No. 330, pp. 1–8, January 1999

REVIEW ARTICLE

New insights into the structure and function of sedoheptulose-1,7-bisphosphatase; an important but neglected Calvin cycle enzyme Christine A. Raines1,3, Julie C. Lloyd1 and Tristan A. Dyer2 1 Department of Biological Sciences, University of Essex, Colchester CO4 3SQ, UK 2 Department of Molecular Genetics, JI Centre, Colney Lane, Norwich NR4 7UJ, UK Received 21 July 1998; Accepted 3 September 1998

Abstract

Key words: Gene expression, photosynthesis, primary carbon metabolism, redox regulation, SBPase, structure, transgenic plants.

Introduction The enzyme sedoheptulose-1,7-bisphosphatase (SBPase) functions in the primary pathway of carbon fixation, the photosynthetic carbon reduction (Calvin) cycle, which in higher plants is located in the chloroplast stroma. The Calvin cycle comprises 11 different enzymes catalysing 13

3 To whom correspondence should be addressed. Fax: +44 1206 873416. E-mail: [email protected] © Oxford University Press 1999

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The photosynthetic carbon reduction (Calvin) cycle is the primary pathway for carbon fixation and the enzyme sedoheptulose-1,7-bisphosphatase functions in the regenerative phase of this cycle where it catalyses the dephosphorylation of sedoheptulose1,7-bisphosphate. This enzyme is unique to the Calvin cycle and has no counterpart in non-photosynthetic organisms. The isolation and sequence analysis of an SBPase clone has led to a number of investigations which have yielded interesting and novel information on this enzyme and in this paper the biochemistry and molecular biology of SBPase are reviewed. Some recent exciting developments are also reported, including the analysis of transgenic plants with reduced levels of SBPase which has shown that SBPase is a key regulator of carbon flux and mutagenesis studies which have resulted in the identification of the redox active cysteines responsible for the regulation by light of SBPase catalytic activity.

reactions and utilizes the products of the light reactions of photosynthesis, ATP and NADPH to fix atmospheric CO into carbon skeletons which are then used directly 2 for starch and sucrose biosynthesis (Fig. 1) ( Woodrow and Berry, 1988; Geiger and Servaites, 1995; Quick and Neuhaus, 1997). This cycle can be considered to have three stages, the first of these being carboxylation of the CO acceptor molecule ribulose-1,5-bisphosphate (RubP) 2 by the enzyme ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco), resulting in the formation of 3-phosphoglycerate. This is followed by the reduction phase which produces the triose phosphates, glyceraldehyde and dihydroxyacetone phosphate, consuming ATP and NADPH in the process. The final phase of the cycle is the regenerative stage when triose phosphates are used to produce the CO acceptor molecule RubP. The carb2 oxylation and reduction steps of the cycle can be considered together as the linear assimilatory phase, the products of which are available for allocation either to RubP regeneration within the cycle or to the starch and sucrose biosynthetic pathways. It is extremely important that a balance is maintained between the amount of carbon leaving the cycle and that needed to maintain RubP levels for the continued functioning of the cycle. In order to achieve this balance the catalytic activities of certain enzymes within the cycle are highly regulated. In particular, a number of the enzymes, including SBPase, are subject to redox regulation via the ferredoxin/thioredoxin system which modulates their activity in response to light conditions (Buchanan, 1980, 1992; Scheibe, 1991; Jacquot et al., 1997a). The regulatory properties of SBPase together with its position at the branch point between the assimilatory and regenerative stages of the cycle have

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highlighted it as one of the enzymes which contribute to the control of Calvin cycle carbon flux. This review focuses on recent developments in the biochemistry and molecular physiology of this previously neglected but important enzyme.

Biochemical studies SBPase ( EC 3.1.3.37) catalyses the dephosphorylation of sedoheptulose-1,7-bisphosphate (sed-1,7-bP) to sedoheptulose-7-phosphate (sed-7-P). This enzyme is unique to the Calvin cycle and unlike many of the other enzymes of this cycle has no cytosolic counterpart. SBPase is one of the two bisphosphatase enzymes in the Calvin cycle, the other being fructose-1,6-bisphosphatase (FBPase). SBPase is found only in photosynthetic organisms and, perhaps for this reason, it has been neglected in comparison with FBPase. In the 1960s and 1970s it was thought that a single enzyme catalysed both chloroplast bisphosphatase reactions, as no distinct SBPase enzyme had been isolated (Buchanan et al., 1976). Indeed, in some photosynthetic bacteria, Rhodobacter and Alcaligenes, ‘FBP’ enzymes with dual function are known, catalysing the dephosphorylation of both sed-1,7-bP and fructose-1,6-bisphosphate (Gerbling et al., 1986). However, in 1978, Breazeale and colleagues identified an enzyme with substrate specificity for sed-1,7-bP in spinach

(Breazeale et al., 1978). Subsequently, SBPase was purified to homogeneity from maize (Nishizawa and Buchanan, 1981) and later from spinach (Cadet et al., 1987) establishing that, in higher plants, SBPase was a distinct enzyme. These studies revealed that the SBPase enzyme is homodimeric, comprising two identical subunits of molecular weight 35–38 000 and that it is immunologically distinct from FBPase (Cadet and Meunier, 1988a). In common with several Calvin cycle enzymes SBPase is virtually inactive in the dark, but within 15 min of illumination activity increases between 15–30-fold, dependent on the plant species (Laing et al., 1981; Wirtz et al., 1982). Light activation of SBPase is mediated through reducing power produced by the photosynthetic light reactions (Fig. 2). This reducing power is transferred via ferredoxin and thioredoxin f to a disulphide bond in the SBPase protein (see below) (Buchanan, 1980). Thioredoxin is thought to bind to the target enzyme forming a stable complex (Geck et al., 1996; Jaramillo et al., 1997) prior to the stepwise reduction of the SBPase disulphide bond. This involves the formation of protein– protein mixed disulphide bonds followed by the release of the reduced SBPase protein and the formation of oxidized thioredoxin (Brandes et al., 1996). This changes the conformation of the active site resulting in activation of SBPase. Light regulation of Calvin cycle enzyme activity was considered only to act as a simple on/off


Fig. 1. The Calvin cycle showing the steps from carboxylation, catalysed by the enzyme Rubisco, to regeneration of the acceptor molecule ribulose-1,5-bisphosphate. The four light-activated enzymes of the cycle are shown in bold.

Molecular biology of SBPase

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Fig. 2. Schematic diagram of the ferredoxin/thioredoxin-mediated light regulation of SBPase activity. Light energy drives electrons through the photosynthetic electron transport chain to the acceptor molecule in photosystem I (PSI ). Electrons from PSI then reduce ferredoxin which in turn brings about the reduction of thioredoxin f, via the enzyme ferredoxin/thioredoxin reductase. Finally thioredoxin activates the SBPase enzyme by reducing the disulphide bridge between Cys-52 and Cys-57, forming two thiol groups.

Molecular biology The isolation and nucleotide sequencing of cDNA and genomic clones coding for the wheat and Arabidopsis SBPase enzymes have been described (Raines et al., 1992; Willingham et al., 1994). More recently additional SBPase sequences from spinach (Martin et al., 1996) and Chlamydomonas (Hahn et al., 1998) have been reported. SBPase is encoded in the nuclear genome and, as a consequence, is synthesized in the cytosol as a precursor protein with an N-terminal extension, known as the

transit peptide, which directs the enzyme to the chloroplast. The derived amino acid sequences obtained from both the wheat and Arabidopsis SBPase genes predict precursor proteins of 393 amino acids. A comparison of these two higher plant sequences revealed that they are 79% identical and if conservative changes are taken into account the similarity rises to 86%. Extending the comparisons to include the Chlamydomonas sequence showed that even this algal sequence has around 73% identity with SBPase from wheat, spinach and Arabidopsis. At present no data are available either from import studies or from N-terminal peptide sequencing of the mature protein to allow identification of the transit peptide cleavage site. However, a putative cleavage site has been proposed based on comparison of the Arabidopsis and wheat deduced SBPase amino acid sequences and the presence of characteristic chloroplast transit peptide motifs in the N-terminal regions ( Willingham et al., 1994). If the putative transit sequences are excluded then comparison of the mature proteins reveals an even closer relationship of 98% similarity in higher plants ( Willingham et al., 1994) with a predicted molecular mass in the range of 35–36 kDa, in agreement with earlier biochemical studies (Nishizawa and Buchanan, 1981; Cadet et al., 1987).

SBPase structure Given the close functional relationship between the stromal bisphosphatases it was of interest to use the sequence information to question whether structural similarities could be found between SBPase and FBPase. It was clear from initial sequence comparisons, of SBPase and FBPases from diverse sources, that these two enzymes are structur-


switch to prevent futile cycling of carbon in the dark. However, it is now thought that thiol regulation of the Calvin cycle also acts to modulate enzyme activity in response to more subtle alterations in the light environment, such as shading and sunflecks (Scheibe, 1991; Jacquot et al., 1997a). SBPase activity is also regulated by stromal pH and Mg2+ levels, both of which change in response to light/dark transitions, indeed the substrate for SBPase is sed-1,7-bP-Mg2+ (Portis et al., 1977; Purczeld et al., 1978; Nishizawa and Buchanan, 1981; Woodrow and Walker, 1982; Woodrow et al., 1984; Cadet and Meunier, 1988b). An additional level of control is also exerted by the products of the SBPase reaction, inorganic phosphate and sed-7-P (Schimkat et al., 1990). SBPase may also be subject to a further regulation as there is some evidence to suggest that it may be part of a functional multienzyme complex. Immuno-electron microscopy has shown that SBPase is localized on the thylakoid membrane in association with ribulose-5-phosphate kinase (PRKase), glyceradehyde-3-phosphate dehydrogenase (GAPDH ) and the electron acceptor ferredoxin reductase (Suss et al., 1993).

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Raines et al. SBPase reaction is a sed-7-bP-Mg2+ complex (Cadet and Meunier, 1988b). One interesting observation from the sequence comparison of the chloroplastic bisphosphatases is the absence of conservation of the redox active cysteine residues involved in thioredoxin regulation of enzyme activity. In chloroplastic FBPase a variable loop region containing three cysteine residues, not found in mammalian, yeast, bacterial or plant cytosolic FBPases, has been identified as the thioredoxin regulatory site (Raines et al., 1988; Marcus et al., 1988; Jacquot et al., 1995, 1997b). Despite the similarities between FBPase and SBPase in overall structure this sequence motif is not found in the SBPase protein. Attempts have been made, using modelling, to deduce which cysteines in SBPase are responsible for thiol regulation (Li et al., 1994; Anderson et al., 1996). However, the results of this approach have been inconclusive as some regions of the SBPase structure could not be modelled with sufficient accuracy. More recently, a mutagenesis approach using a wheat cDNA clone (Raines et al., 1992) has revealed that the residues C52 and C57 are likely to be the regulatory cysteines in SBPase. Mutation of either of these residues to serines results in an active but redox-insensitive enzyme (Dunford et al., 1998). C52 and C57 are located in a flexible loop near to the junction between the subunits ( Fig. 2) therefore making or breaking of a disulphide bond between these cysteines could alter the conformation of the dimer and lead to changes in activity. To confirm these findings it will be necessary to show that these residues are linked to one another only when the enzyme is in the oxidized (inactive) form. The next step in the elucidation of the relationship between the structure and function of SBPase will require large scale purification and crystallization of this protein. For example, such studies will be needed to establish the structural differences between the oxidized and reduced forms of the enzyme which will lead to an understanding of the structural details of the allosteric changes which occur during redox regulation. The information now available from mutagenesis studies has revealed that the regulatory cysteine residues in the FBPase and SBPase protein sequences are located in different positions. The feature that they have in common is that the redox active cysteines are distant from the catalytic site. This is in contrast with PRKase where the cysteines involved in thiol regulation are some 39 amino acids apart and are located within the active site region of this protein. This work suggests that in each case thiol regulation has evolved independently in response to the appearance of oxygenic photosynthesis (Buchanan, 1991; reviewed in Jacquot et al., 1997a).

Regulation of SBPase gene expression The expression of the gene encoding SBPase, like other nuclear encoded Calvin cycle enzymes is affected by light,


ally related. Between 29–32% of residues are identical and if conservative changes are included this rises to 52% similarity (Raines et al., 1992). Recently, a more extensive phylogenetic study using additional SBPase sequences was carried out which indicated that FBPase and SBPase share a common evolutionary origin. This study suggested that these two enzymes acquired substrate specificity following divergence from a bifunctional enzyme (Martin et al., 1996; Martin and Schnarrenberger, 1997). The conservation of amino acid sequence in the SBPase and FBPase proteins was found to be greater in some regions than others, reflecting the important functional role of these amino acids (Raines et al., 1992). Confirmation of this has come from crystallographic work on the 3D structure of FBPase which has identified amino acids in the active site region of this enzyme ( Ke et al., 1989, 1990a, b; Zhang et al., 1994; Villeret et al., 1995). The conserved regions common to FBPase and SBPase identified by the amino acid comparisons contain the residues implicated in binding of the FBPase substrate, a finding that suggests conservation of the active site structure of the two bisphosphatase enzymes ( Ke et al., 1991). Given their common evolutionary origin and the similarity of their substrates it is not surprising to find that of the 19 amino acids implicated in substrate binding in FBPase, 12 are also conserved in SBPase (Raines et al., 1992). In particular, the amino acids involved in binding the 1-phosphate and the carbohydrate backbone of each substrate (fru-1,6-P, sed-1,7-P) are highly conserved in all the SBPases and FBPases sequenced to date. In contrast, the amino acids identified as the likely 6- and 7-phosphate ligands are not conserved and this is probably the basis of the different sugar specificities of the two enzymes ( Raines et al., 1992). The structural similarities between SBPase and FBPase highlighted by these amino acid sequence comparisons enable the use of the 3D structure of FBPase as a model to gain an insight into the structure of SBPase. Crystallographic studies of pig (Zhang et al., 1994) and spinach chloroplast ( Villeret et al., 1995) FBPase show that each subunit is composed of a series of alpha helices and beta sheets joined by turns and loops and folded into a hexahedron shape. The SBPase sequence may be folded in the same way with a high degree of confidence, especially with respect to the helix predictions. Each subunit is paired forming a functional dimer (in contrast to FBPase which is a tetramer consisting of two dimer pairs) with an active site on the interface between the subunits. In FBPase the sidearm of an arginine residue from the adjoining subunit is necessary to complete each active site. A magnesium ion located at the active site takes part in the catalytic reaction and changes in the localization of this ion are thought to regulate FBPase activity ( Zhang et al., 1994). It is likely that this is also the case for SBPase, given that the substrate of the


(Gilmartin et al., 1990). Much less is understood of the sequences and DNA binding proteins involved in regulating gene expression in monocot species. Using in vitro DNA binding assays a site-specific DNA binding protein, WF1, present in wheat nuclei has been identified which interacts with several wheat Calvin cycle genes, including that of SBPase (Miles et al., 1993). More recently the binding site for WF1 has been localized to a region within the promoter of the wheat SBPase gene containing two ACGT motifs. ACGT is the core sequence of a prominent group of cis-acting elements common to many plant promoters which bind the bZIP class of transcription factors ( Katagiri and Chua, 1992). The upstream region of the Chlamydomonas reinhardtii SBPase gene also contains sequences involved in controlling the light-regulated expression of this gene. This algal upstream sequence has been found to confer light-regulated expression on a reporter gene, but the cis-acting elements involved have not as yet been identified (Hahn et al., 1998). Comparison of the wheat and Arabidopsis SBPase genes reveals that they have a similar intron/exon structure; both contain seven introns of which six are located at identical positions in the two genes ( Willingham et al., 1994). In contrast, the algal gene structure exhibits no conservation of intron number or position when compared with the higher plant SBPase genes (Hahn et al., 1998). The Arabidopsis SBPase gene is present as a single copy sequence as is the case in Chlamydomonas ( Willingham et al., 1994; Hahn et al., 1998). In hexaploid wheat five sequences with homology to SBPase are present although only three copies of the gene would be expected, suggesting that gene duplication has occurred in the evolution of this crop species (Devos et al., 1992).

Antisense SBPase in transgenic tobacco Biochemical studies have shown that SBPase activity is highly regulated in vivo and this led to suggestions, supported by modelling studies, that this enzyme may also have some control of the rate of flux of carbon through the Calvin cycle (Breazeale et al., 1978; Wirtz et al., 1982; Woodrow, 1986; Petterson and RydePetterson, 1989; Schimkat et al., 1990). To investigate the relative importance of SBPase, an antisense approach has been used to manipulate the actvity of SBPase in transgenic tobacco plants. The results from this work have shown clearly that a decrease in SBPase activity results in a significant reduction in the rate of light- and CO 2 saturated photosynthesis (Harrison et al., 1998). Even in plants with only a 35% reduction in SBPase activity the photosynthetic capacity was reduced, and flux control coefficient values for photosynthesis from 0.55 to 0.7 were obtained. In contrast, studies using transgenic plants with reduced levels of three other highly regulated Calvin cycle enzymes, FBPase, GAPDH and PRKase showed little


development and levels of hexose sugars (Miles et al., 1993; Willingham et al., 1994; Jones et al., 1996). In dark-grown wheat and Arabidopsis seedlings the level of SBPase mRNA is very low and in the latter is below the level of detection. In both species exposure of dark-grown seedlings to light increased the level of SBPase mRNA by at least 20-fold, with a more rapid response apparent in wheat than Arabidopsis ( Willingham et al., 1994). This may reflect the fact that the early stages of chloroplast development can proceed in the absence of light in monocots and as a consequence they are poised to respond rapidly to the light signal (Mullet, 1988). In studies using the primary wheat leaf Calvin cycle enzyme mRNA levels, including SBPase mRNA, were found to vary considerably during leaf development. Very low levels were detected in cells at the base of the leaf, which contain immature plastids, and these levels increased significantly (5–20-fold ) in the mid-section where the cells are fully expanded and the chloroplasts mature. Towards the leaf tip SBPase and other Calvin cycle enzyme mRNA levels decline, since less mRNA is required to maintain protein concentrations in the mature chloroplast than is needed during development of the chloroplast (Raines et al., 1991; Willingham et al., 1994). Recent studies have suggested that, in addition to developmental regulation, this reduction in Calvin cycle mRNA levels may result from metabolic feedback on gene expression. In support of this, it has been shown that feeding glucose to intact wheat plants leads to a reduction in Calvin cycle mRNAs, including that of SBPase but only in developmentally mature tissue (Jones et al., 1996). Immunolocalization and in situ hybridization techniques have been used to study the effect of elevated internal glucose concentrations on the levels of chloroplastic FBPase protein and mRNA. The results indicate that there is not only an interaction between developmental stage and metabolic status, but that the metabolic status of the cells may also attenuate the light induction response of Calvin cycle mRNAs (Greene, 1998). The biogenesis of a functional Calvin cycle during chloroplast development requires the co-ordinated synthesis of the eleven enzymes which catalyse the reactions in this complex pathway. It is likely that a significant proportion of the control of this process is at the level of transcription and one possible mechanism for achieving this would be for common nuclear factors to control the transcription of all these genes. The isolation of the nucleotide sequences encompassing the regulatory and promoter regions of the wheat and Arabidopsis SBPase genes has allowed some progress to made towards understanding these mechanisms. The upstream sequence of the Arabidopsis thaliana SBPase gene ( Willingham et al., 1994) contains two GATA motifs with homology to the I box, a conserved DNA sequence element found in the promoter regions of several light-regulated genes

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Future prospects Cloning of the SBPase gene in the early 1990s has facilitated a diverse range of molecular studies on this enzyme. The achievements made to date have given us an understanding of the structure of this enzyme, including the identification of the cysteine residues involved in redox regulation. The next challenge in this area of SBPase research will be to use crystallographic techniques to resolve, at a molecular level, the 3D structural changes which occur during activation. A detailed understanding of the molecular interactions between SBPase and thioredoxin during the light activation process should follow on from such studies. In addition to these structural studies the use of transgenic technology to manipulate the levels of SBPase activity in plants has highlighted the potential importance of this enzyme in the control of photosynthetic carbon fixation. These results have raised the question of whether plants with increased levels of SBPase could support higher rates of photosynthesis, for example, when plants are grown in elevated CO . Another important question 2 remaining is the role of light activation of individual enzymes, such as SBPase, in controlling the flux of carbon through the Calvin cycle. The availability of fully active, deregulated mutants of both SBPase (Dunford et al., 1998) and FBPase (Jacquot et al., 1995, 1997b) produced using site-directed mutagenesis should allow this question

to be addressed in vivo. The continued use of transgenic technology is likely to lead to further significant advances in the understanding of the role of SBPase in Calvin cycle metabolism.

Acknowledgements This work was supported by grants from the Biotechnology and Biological Sciences Research Council and the University of Essex Research Promotion Fund. We would like to thank Alistair Rogers for the Calvin cycle diagram and Minnie O’Farrell for critical reading of this manuscript.

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effect on photosynthetic carbon assimilation until activities were reduced to below 35% of wild-type levels ( Koßman et al., 1994; Price et al., 1995; Paul et al., 1995; reviewed in Stitt and Sonnewald, 1995). Similar studies using Rubisco small subunit antisense plants showed that this enzyme only had a significant flux control co-efficient for photosynthesis in extremely high light (in excess of 1500 (mol m−2 s−1) or in low nitrogen conditions ( Hudson et al., 1992; reviewed in Stitt and Schulze, 1994). Results from experiments in which the SBPase antisense plants were grown in limiting phosphate indicated that SBPase has very little control of photosynthesis in these conditions. To date much of the analysis of photosynthetic capacity in antisense Calvin cycle plants has focused on mature fully expanded leaves. Analysis of the antisense SBPase plants has been extended to include younger developing leaves. The results from this were very interesting and a significant developmental effect on the control of photosynthesis by SBPase has been observed. Flux control coefficients for photosynthesis increased from very low values, 0.15, in young expanding leaves to values of between 0.55 and 0.7 in fully expanded mature leaves (Olcer, 1998). These results provide further evidence that the regulatory capacity of Calvin cycle enzymes in vivo is not constant and varies during development and in response to environmental conditions


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