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Home , Biochemistry, Glutamate synthase, Photorespiration, Roland Douce

214

Biochemical dissection of Roland Douce* and Michel Neuburger

Progress has been made in the understanding of each other. In the course of this pathway two of photorespiration and related (Rubisco, glycolate glycolate-2-P are metabolized to form one each of oxidase and decarboxylase) in the context of recent glycerate-3-P and CO2 and these compounds are structural information. Numerous shuttles exist to support used immediately for the regeneration of RuBP via the , refixation and the supply or export of Benson– (C3 cycle) without the net synthesis reductants generated or consumed (via malate-oxaloacetate of . Once glycolate-2-P is formed, the pho- shuttles) in the photorespiratory pathway. A porin-like channel torespiratory cycle works forward to convert all the carbon that is anion selective represents the major permeability diverted out of the C3 cycle back to as pathway of the peroxisomal membrane. rapidly as possible [3]. Indeed, several reactions occuring in and strongly favor forma- Addresses tion. Obviously, although very little is known about the DBMS, Laboratoire de Physiologie Cellulaire Végétale, feed-back mechanisms that might operate in photorespira- CEA et Université Joseph Fourier, 17 rue des martyrs, tion [4], the most important control step is at the level of F 38054 Grenoble, Cedex 9, France competition between O and CO for binding to Rubisco. *e-mail: [email protected] 2 2

Current Opinion in 1999, 2:214–222 In C3 the C2 cycle is operating in the photosynthet- http://biomednet.com/elecref/1369526600200214 ically active mesophyll cells. In C4 plants the C2 cycle operates in the bundle sheath cells [5]. Using two geneti- © Elsevier Ltd ISSN 1369-5266 cally modified C4 plants, a of Amaranthus edulis that Abbreviation is deficient in PEP carboxylase and a transgenic plant RuBP ribulose-1,5-bisphosphate Flaveria bidentis which has reduced levels of Rubisco, • Marocco et al [6 ] have demonstrated that when the C4 Introduction plant is ineffective in concentrating CO2 in the bundle The prime of the C2 oxidative photosynthetic car- sheath cells there is a marked increase in photorespiration bon cycle — inappropriately named ‘photorespiration’ and when the C4 plant exhibits low levels of Rubisco there • [1 ] — is to salvage glycolate-2-P produced continuously in is a marked increase in bundle-sheath CO2 leakage. This the light by the activity of ribulose-1,5-bisphos- observation provides definitive evidence that photorespi- phate carboxylase/oxygenase (Rubisco). In leaves under ration is insignifiant in C4 plants because they are capable ambient conditions the rate of oxygenation to of concentrating CO2 in the bundle-sheath cells leading to has been estimated as high as 0.4. Low intercellular con- the suppression of the oxygenase reaction of Rubisco. centrations of CO2, as may occur, for example, under (e.g. whenever the stomata are closed), can result in Functioning of key involved in even higher ratios. Given the voluminous literature on pho- photorespiration torespiration [2,3], in this short review we merely highlight Few enzymes involved in this cycle have been studied recent advances in this topic, laying emphasis on a few pho- carefully. Only Rubisco, glycolate oxidase and a sophisti- torespiratory enzymes (Rubisco, glycolate oxidase and cated set of proteins involved in glycine cleavage (glycine glycine decarboxylase) and molecular traffic between per- decarboxylase system) have been studied in an exhaustive oxisomes, chloroplasts, and mitochondria. manner. For this reason we have chosen to focus on a restricted set of systems. The photorespiratory pathway The value of numerous mutant plants (Hordeum, pea, and Mechanism of Rubisco: the triggering of photorespiration Arabidopsis thaliana) in the exquisite elucidation of the mech- Rubisco is present at a tremendous concentration in the –1 anism of photorespiration and its relationships with CO2 stroma of the chloroplasts (~0.2 g ml ) stromal extract and fixation and has been highlighted by catalyses both the carboxylation (the enzyme exhibits a low several groups (see [3] for a full list). These were catalytic rate constant, 3.5 sec–1) and the oxygenation of unable to survive in air, but could thrive in atmospheres con- ribulose-1,5-bisphosphate [7–9,10••]. The two reactions taining a high concentration of CO2 (or low [O2]). involve the competition of molecular CO2 with O2 for the 2,3-enediol(ate) form of RuBP which is first generated at the The recycling of glycolate-2-P into glycerate-3-P via the active site of the enzyme. At any given [CO2][O2], the frac- photorespiratory pathway and then further to ribulose-1,5- tional partitioning of RuBP between the carboxylation and bisphosphate (RuBP) is not only a very costly reaction, it oxygenation pathways is governed by the relative reactivity also requires a large machinery consisting of 16 enzymes of the enzyme-bound 2,3-enediol(ate) toward CO2 and •• and more than six translocators, distributed over the chloro- O2 [10 ]. From biochemical analyses of Rubisco purified plast, and in close proximity to from several species, including photosynthetic , Biochemical dissection of photorespiration Douce and Neuburger 215

, green , and higher plants, there are large The oxygenation of RuBP yields one molecule each of differences in specificity towards the substrates CO2 and O2: glycerate-3-P (formed from C-3, C-4 and C-5 of RuBP), evolutionary pressures seem to have directed Rubisco and glycolate-2-P. The oxygenation pathway has not been •• •• towards more efficient utilization of CO2 [10 ,11]. Rubisco dissected as deeply as the carboxylation counterpart [10 ]. from cyanobacteria, green algae and higher plants is assem- Very likely the oxygenation pathway is similar to the car- bled from eight large (L) subunits and eight small (S) boxylation pathway although the putative key labile subunits (four dimers of L subunits surrounded by two intermediate (2-peroxy-3-ketoarabinitol 1,5-bisphosphate) •• tetramers of S subunits; (L2)4(S4)2) [12], whereas Rubisco [10 ] postulated through the exquisite characterization of → → from Rhodospirillum rubrum (a nonsulfur purple bacteria) two-different site directed mutants (E60 Q, K334 A) consists of only two large subunits (L2). The large subunit [18,19], has never been characterized so far. This reaction from spinach can be divided into two domains, an amino may be an inevitable consequence of Rubisco’s inability to α β terminal and a carboxy-terminal / -barrel domain. protect its ene-diolate reaction intermediate from O2. Two active sites are located at the interface of the L-sub- Indeed, this notion is supported by the failure of numerous units in the L2 dimer (‘head to tail’ arrangement). The efforts to eliminate selectively its oxygenase activity by catalytic center is mostly situated at the carboxy-terminal genetic manipulation. The partitioning of RuBP between end of the α/β-barrel. The enhancement of catalytic rate by the carboxylation and oxygenation pathways is sensitive to S subunits can only mediated through induced conforma- the active site microenvironment and does not involve tional changes in catalytic subunits because S subunits are large movements within the structure [10••]. Given the far removed from the active site [13]. structural similarity of the two alternative substrates CO2 and O2 and the large difference in their concentration A large number of crystal structures of Rubiscos from var- within the chloroplasts, it is clear that Rubisco influences ious sources including Rhodospirillum rubrum, the selectivity for CO2 in some way [12]. Synechococcus, and spinach have been reported along with a variety of ligands (see [10••,], for a full list) and this, in Glycolate oxidase synergy with biochemical investigations, led to a careful Glycolate oxidase (an octamer composed of identical sub- dissection of the carboxylation pathway. The carboxyla- units of approximately 40 kDa) is one of the very few tion of RuBP involves multiple discrete steps and peroxisomal proteins for which a high resolution crystal associated transition states: removal of ligands such as 2- structure is available [20•]. The enzyme from spinach crys- carboxyarabinitol 1-phosphate from the inactive enzyme tallizes in an octameric form and the subunit contains an form (this process occurs slowly by simple , or eight-fold β/α barrel motif corresponding to the flavin rapidly when catalysed by the enzyme Rubisco activase); mononucleotide (FMN) domain which is also found in carbamylation of the ε-amino group of Lys201 (spinach) other FMN-dependent enzymes. The irreversible reaction in the active site by an activator CO2 molecule; catalysed by the enzyme can be divided into two half-reac- stabilization of this -bound carbamate by mon- tions. First glycolate is oxidised by the flavin which is odentate coordination to Mg2+ (three water molecules, deeply burried in the barrel. In the second part FMN is Asp203 and Glu204 complete the octahedral coordination reoxidized by O2 to produce H2O2 which is, in turn, sphere around this ); binding of RuBP: it is ori- decomposed by (a -containing enzyme). The ented in the active site with the Si face of the C-2 (and active site is formed by the loops at the carboxy-terminal C-3) accesible to the bulk solution (for an explanation, end of the β-strands in the barrel. The amino acids see [10••]); removal of the C-3 of RuBP to effect involved in the structure of the active site have been stud- enolisation (the deprotonating agent is still not identi- ied [21]. Thus, the replacement of Trp108 by Ser led to fied); addition of CO2 to the Si face of C-2 and water to dramatic effects on both the Km of as well as on the Si face of C-3 to yield the six-carbon hydrated inter- the turnover number indicating that this amino acid is of mediate (2′-carboxy-3-keto-D-arabinitol 1,5 crucial importance in and in determining the sub- bisphosphate); and carbon–carbon cleavage between C-2 strate specificity of glycolate oxidase. Likewise Tyr24 is and C-3 to form two glycerate-3-P molecules] [10••,14••]. involved in binding of the substrate by way of - Higher resolution structures of both the Synecococchus [15] bond formation between its hydroxyl group and the and of the spinach enzyme [12,14••] demonstrated the carboxylate group of the substrate molecule. key role of the carbamate on K201 in the carboxylation pathway. The role of Rubisco activase in limiting steady The uptake of glycolate oxidase into peroxisomes has been state photosynthesis has been examined using transgenic studied [22]. The signal for targeting glycolate oxidase into plants with reduced levels of activase [16,17]. It was con- the plant peroxisome is rather complex. Apparently the cluded that Arabidopsis grown under high and low amino-terminal 59 amino acids are dispensible for protein irradiance does not contain Rubisco activase in great import in an ATP-dependent and temperature-dependent excess of the amount required for optimal growth [16]. In manner. This raises the question of the presence of a addition, a phase in the of Rubisco that repre- carboxy-terminal hexapeptide (RAVARL) at the carboxy- sents the activation of the 2-carboxy arabinitol 1 terminus of the protein which plays also a role in targeting phosphate inhibited form of Rubisco was discerned [17]. a protein to peroxisomes. 216 and metabolism

Figure 1

Outline of the reactions involved in oxidative S decarboxylation and deamination of glycine in plant mitochondria. Glycine decarboxylase S consists of four different component proteins: P, T, H, and L. H-protein is a 14.1 kDa O that plays a pivotal role in the reaction mechanism, as it interacts sequentially COO H N with each of the other three proteins through H 2C its bound to a residue. The P-protein component (this NH3 NADH enzyme has a Mr of 210,000 and is a homodimer of 105,000 Mr polypeptides) catalyses the decarboxylation of glycine and Hox the reductive transfer of the resultant P methylamine moiety to the lipoyl-lysine CO L 2 NAD (lipoamide arm) of the H-protein. The lipoate cofactor is located in the loop of a hairpin configuration, but following methylamine transfer, it is pivoted to bind into a cleft at the H H surface of the H-protein. The lipoamide- met red methylamine arm is, therefore, not free to move O in the solvent. The lipoamide-methylamine arm NH3 NH 3 is then shuttled to the T-protein (a 45,000 M O NH CH NH r 2 monomer) where the methylene carbon is S transferred to tetrahydrofolate (H4FGlu5), SH producing CH2-H4FGlu5 and releasing the HS amino as NH3. Finally, the reduced lipoamide resulting from this transfer is HS T reoxidized by the FAD coenzyme bound to the L-protein (a homodimer of 60,000 Mr polypeptides), with the sequential reduction of H FGlu CH H FGlu 4 5 2 4 5 FAD and NAD+. SHMT, hydroxymethyltransferase is involved in the COO COO recycling of CH2-H4FGlu5 to H4FGlu5. HOH C H C H 2C 2 2 NH NH3 3 SHMT Current Opinion in Plant Biology

During the course of glycolate oxidation, proceeding in an plant mitochondria, has been purified and, like its mam- irreversible way, huge amounts of are malian counterpart, contains four different component released in the peroxisomes. Most of the hydrogen perox- enzymes designated as the H-protein (a monomeric ide is degraded by catalase, but the high Km (millimolar lipoamide-containing protein, 14 kDa), P-protein (a range) for the enzyme could result in low harmful residual homodimer containing pyridoxal phosphate, 200 kDa), T- concentrations diffusing into contact with the inner surface protein (a monomer acting in concert with of the limiting peroxisomal membrane which contains an [5,6,7,8-tetrahydropteroylpolyglutamate; H4PteGlu], ascorbate peroxidase [23]. Transgenic tobacco with 0.05 to 45 kDa) and L-protein or lipoamide dehydrogenase (a 0.15 times the catalase activity of wild-type has been homodimer containing flavin dinucleotide [FAD] reported [24], and it was shown that under high photores- and a redox active cystine residue, 100 kDa) [25]. All the piratory conditions necrotic lesions were produced in protein components of the glycine decarboxylase system leaves owing to dramatic accumulation of H2O2. dissociate very easily and behave as non-associated pro- teins following mitochondrial inner membrane rupture Reaction catalysed by the glycine after several cycles of freezing and thawing. decarboxylase multienzyme complex Rapid glycine oxidation, which requires the functioning of The H-protein acts as a mobile co-substrate that com- two enzymatic complexes (glycine decarboxylase and ser- mutes between the other three proteins (Figure 1). Its ine hydroxymethyltransferase) working in concert, is a lipoyl moiety (attached by an amide linkage to the ε-amino key step of the C2 cycle because it results in the conver- group of a lysine residue [Lys63 in the 131 amino acid pea sion of a two-carbon molecule into a three-carbon H-protein; 26] which is located in the loop of an hairpin molecule that thereafter, could be reintroduced in the C3 configuration [27]) undergoes a cycle of reductive methy- cycle [25]. The glycine decarboxylase multienzyme com- lamination, methylamine transfer and electron transfer. plex, present at tremendous concentration in the matrix of The reaction commences with the amino group of glycine Biochemical dissection of photorespiration Douce and Neuburger 217

forming a Schiff with the pyridoxal phosphate of the synthesis and control of the distribution of this unique P-protein. The carboxyl group of glycine is lost as CO2 and enzyme associated with different complexes. For example, the remaining methylamine moiety is passed to the the distribution of L-protein among complexes may rely lipoamide cofactor of the H-protein; when it is oxidized upon various metabolic situations. the lipoyl moiety is free to move in the solvent and is allowed to visit the active site of the P-protein. The rapid In leaf mitochondria, the major function of serine hydrox- methylamination of the H-protein is half-saturated at ymethyltransferase (SHMT, a 220 kDa homotetramer) is micromolar concentrations of H-protein (Km H-protein to recycle CH2H4PteGlun produced by the T-protein activ- µ µ –1 –1 =9 M; Vmax =5 mol mg protein min ). During the ity to H4PteGlun, to allow the continuous operation of the course of the reductive methylamination, the lipoamide- glycine-oxidation reaction [31]. This reaction is perma- methylamine arm formed rotates to interact readily with nently pushed out of equilibrium towards the production several specific amino acid residues located within a cleft of serine and CH2H4PteGlun [32] that is the forward at the surface of the H-protein; the methylamine group motion of the photorespiratory cycle. The T-protein and linked to the distal of the dithiolane ring is tightly SHMT do not associate and the reaction intermediates are bound by ionic and hydrogen bonds to residues Glu14, not directly transferred through a channeling mechanism Ser12, and Asp67, whereas the carbon of the from the active site of T-protein to that of SHMT. lipoamide arm interact through van der Waals contacts with hydrophobic residues [27,28]. Photorespiratory nitrogen cycle Quantitatively, the conversion of glycine to serine in the Such a situation locks the methylamine group into a very C2 cycle is probably the most important metabolic process stable conformation preventing the non-enzymatic release that liberates ammonia within the mesophyll cells. of NH3 and formaldehyde which would otherwise take Nitrogen is inserted into the C2 cycle through a transami- place due to nucleophilic attack by OH– of the carbon nation step in the peroxisome catalysed by a bearing NH2 group until the reaction with glutamate:glyoxylate aminotransferase [2]. Ammonia liber- H4PteGlun and T-protein takes place. In the absence of ated in the matrix of mitochondria during the course of H4PteGlun in the incubation medium the T-protein causes glycine oxidation diffuses rapidly to the where a change in the overall conformation of the H-protein, it is used, with a very high affinity, by syn- leading to the release of the lipoamide-methylamine arm thetase catalysing the ATP-dependent conversion of from the cleft at the surface of the H-protein. These cir- glutamate to glutamine [33]. Indeed Mattson et al. [34] cumstances, therefore, favour, the nucleophilic attack by demonstrated that in barley mutants with reduced gluta- – OH of the carbon atom bearing NH2 group; NH3 and mine synthetase the rate of ammonia emission correlated formaldehyde accumulate slowly in the incubation medi- with the concentration of ammonia in the leaves. In bacte- um and the lipoamide arm becomes fully reduced rial , the active site is located (Figure 2). On the other hand, in the presence of between adjacent subunits and structural models for the H4PteGlun formaldehyde does not accumulate because reaction mechanism based on five crystal structures of the methylamine group undergoes a preferential nucle- enzyme–substrate complexes have shown that the reaction ophilic attack by the N-5 atom of the pterin ring of occurs in two steps. First ATP binds to the active site fol- γ H4PteGlun: NH3 and CH2H4PteGlun; accumulate rapidly lowed by glutamate to yield -glutamyl phosphate and + in the medium concomitantly with the reduction of the ADP. Then NH4 binds to the active site, which, after los- lipoamide arm (Figure 2). ing a proton, attacks the γ-glutamyl phosphate with the liberation of glutamine and phosphate [35,36]. Whether a Plant mitochondria possess a powerful NAD-dependent for- similar mechanism also operates in eukaryotic octameric mate dehydrogenase [29]. They also possess a formaldehyde glutamine synthetase is still a matter of debate. It is clear dehydrogenase. These enzymes are not believed to be now from the analysis of barley mutants deficient in gluta- involved in the main route of carbon flow through the glyco- mine synthetase that the chloroplastic isoform is directly late pathway. They could serve as rescue reactions, involved in the reassimilation of ammonia released during neutralising the harmful effect of formaldehyde molecules the process of photorespiration [33]. On the other hand, produced by the in a non-controlled the cytoplasmic isoform is localized in the vascular system reaction. Finally, the L-protein (dihydrolipoamide dehydro- and the phloem companion cells of the leaf [37,38], thus genase) catalyses the regeneration of the oxidised form of precluding any role in photorespiration. lipoamide with the sequential reduction of FAD and NAD+. This rapid oxidation is half-saturated at micromolar concen- Ferredoxin-dependent glutamate which is exclu- trations of H-protein (Km reduced H-protein = 20 µM). In sively localised in the chloroplast of mesophyll cells green leaf mitochondria, the pyruvate dehydrogenase and catalyses the reductant-dependent conversion of gluta- glycine decarboxylase complexes share the same dihy- mine and 2-oxoglutarate to two molecules of glutamate. drolipoamide dehydrogenase (E3 component of pyruvate This enzyme, therefore, functions coordinately with gluta- dehydrogenase, L-protein of glycine decarboxylase) [30] and mine synthetase. One molecule of glutamate thus formed this raises some interesting questions about the regulation of is exported to the peroxisomes as an amino donor for 218 Physiology and metabolism

Figure 2

(a) H H H H HN HN HN HN O O O O

NH HS 3 HS HCHO H HS HS H O CH S S HS CH S 2 NH3 2

OH

(b) HFGlu 4 n CH H F Glu H 2 4 n 5 H H N H H 10 N N H NH R N N NH R NH R H H C NH R H NH R 2 H C H NCH S CH S CH S 2 HS 3 2 2 2 S HS HS HS HS HS H NH3

O O O O O NH NH NH NH NH H H H H H

Current Opinion in Plant Biology

Proposed model for the reaction catalysed by the T-protein. (a) In the NH3 and formaldehyde accumulate slowly in the incubation medium absence of H4FGlun in the incubation medium the T-protein causes a and the lipoamide arm becomes fully reduced. (b) On the other hand, change in the overall conformation of the H-protein, leading to the in the presence of H4FGlun, the methylamine group undergoes a release of the lipoamide-methylamine arm from the cleft at the surface preferential nucleophilic attack by the N-5 atom of the pterin ring of of the H-protein (see Figure 1). Such a situation favours, therefore, the H4FGlun. CH2H4FGlun is, therefore, rapidly formed in place of – nucleophilic attack by OH of the carbon atom bearing the NH2 group; formaldehyde, concomitantly with the reduction of the lipoamide arm. glutamate:glyoxylate amino transferase in exchange for Molecular traffic 2-oxoglutarate. Arabidopsis contains two expressed During the course of photorespiration, massive traffic of for this enzyme (Glu1 and Glu2) situated on different various molecules occurs between different organelles. chromosomes. Glu1 plays a major role in photorespiration Unfortunately, the major characteristics of the transport in Arabidopsis, as has been determined by the characteriza- proteins (reconstitution of the transporter into , tion of mutants deficient in this form [39]. Glu2 may play kinetic parameters, multisubunit , high-resolution a major role in primary nitrogen in roots. The structures, and multifaceted regulation) catalysing sub- enzyme (monomeric with an Mr of ~160 kDa) contains one strate travel through membranes to fulfil photorespiration FMN and one {3Fe-4S} cluster per molecule [40]. The have been poorly studied. We must say that it is always a of this enzyme activity has been greatly facilitated by real ‘tour de force’ to reconstitute a transporter into lipo- the use of methyl viologen as a source of reductant [41] somes in an active form. which is recognized by the ferredoxin-binding site con- taining two critical lysine and residues [41,42]. NH3 and CO2 movement + The NH4 (and/or NH3) released during glycine oxidation An interesting point recently raised by Migge et al. [43•] passes through the inner membrane of mitochondria and was that key enzymes of the photorespiratory nitrogen chloroplasts. Whether this passage occurs by simple diffu- cycle were not affected either by growing plants in elevat- sion, or is brought about by specific ion channels or ed CO2 partial pressure (short-term exposure) or by the translocators is still a matter of debate. In order to maintain rate of photorespiratory production, thus ammonia emission close to zero when carbon assimilation allowing C2-cycles and nitrogen-cycles to take place imme- is strongly limited by stomatal closure under drought con- diately following exposure to normal air. ditions, we should expect a specific mechanism to divert Biochemical dissection of photorespiration Douce and Neuburger 219

ammonia towards chloroplasts where it is assimilated. In functions as a monomer, in contrast to other known trans- support of this suggestion a from Arabidopsis for a porters of organellar origin, including mitochondria, that high affinity ammonia transporter has been identified [44]. have 5–7 transmembrane helices functioning as dimers. We can speculate, therefore, the presence of a specific The transit peptide of this translocator is extremely long ammonia transporter on the inner membrane of the chloro- although its import characteristics closely resemble those of plast envelope. other inner envelope membrane proteins. The 2-oxoglu- tarate/malate translocator could be functionally expressed Likewise, one of the major unresolved aspects of the inner in the fission Schizosaccharomyces pombe and subse- membranes of mitochondria and chloroplasts in all eukary- quent reconstitution of the recombinant protein in otes concerns the CO2 permeability of the membranes. In liposomes demonstrated definitively that this translocator other words it is not known which carbon inorganic species mediates the exchange of 2-oxoglutarate with malate. - (CO2, HCO3 ) is transported in cell organelles. In this con- Obviously the glutamate/malate carrier, which also plays a nection Rolland et al. [45], using a mutant of Chlamydomonas critical role in the recycling of ammonia during the course reinhardtii, have suggested the existence of a specific pro- of photorespiration, requires an exhaustive study in order to tein within the envelope which promotes inorganic understand precisely the interplay of both carriers working carbon uptake into chloroplasts. Very likely, this protein is in concert. the product of the chloroplast ycb10 gene which has been localized in the inner membrane of the plastid envelope. Mitochondria transporters The disruption of this gene in Chlamydomonas using biolis- The rate of glycine oxidation demands that green leaf mito- tic transformation was correlated with a decrease in chondria support a phenomenal rate of glycine transport –1 –1 CO2-dependent photosynthesis and a reduced affinity of (0.8–1.6 µmol min mg protein). In the course of glycine the CO2 and HCO3-uptake system for their substrates. decarboxylation and deamination, one molecule of serine leaves the mitochondrion and two molecules of glycine are Chloroplast transporters taken up. For the present, we have to admit that the details Glycolate must move from the stroma to the peroxisome of glycine and serine transport in green leaf mitochondria across the inner envelope membrane and D-glycerate must remain a mystery and the question as to whether both go in the opposite direction. with intact glycine and serine are transported by a single protein or by chloroplasts have shown that a single carrier-type trans- two different ones cannot be answered at present. porter is responsible for the movement of both glycolate and D-glycerate across the chloroplast inner envelope The conversion of hydroxypyruvate to glycerate in the per- membrane. This transporter was solubilized by treatment oxisomal matrix requires NADH as reductant. Peroxisomes of the chloroplast inner membrane by cholate and are, therefore, dependent on the supply of reducing equiv- reinserted into artificial vesicles [46] . The glycolate/glyc- alents from the cytoplasmic compartment. On the other erate transporter is interesting because it does not catalyse hand, NADH produced during the course of glycine oxida- a strictly coupled substrate exchange (however, glycolate tion is reoxidized very rapidly by oxaloacetate owing to the and D-glycerate stimulate one another’s transport from the tremendous excess of NAD+-linked malate dehydrogenase opposite side of the membrane); unidirectional influx or in the matrix space. The malate produced from this reac- efflux also occurs as a proton symport or hydroxyl antiport. tion is removed from the mitochondria in exchange for This flexibility allows the amount of glycerate returning to cytosolic oxaloacetate by a specific oxaloacetate transporter. the chloroplasts to be only half that of the glycolate Peroxisomes are supplied, therefore, with reducing equiva- released from the chloroplasts. lents not by direct uptake of NADH but by indirect transfer via this malate–oxaloacetate shuttle [49]. A very During the course of photorespiration, 2-oxoglutarate is powerful phthalonate-sensitive oxaloacetate carrier has massively imported into the chloroplasts, and glutamate, been characterised in all the plant mitochondria isolated so deriving from the glutamine synthetase/ far [50]. This rapid phthalonate-sensitive uptake of oxaloac- cycle, is exported towards the peroxisome. Two different etate, which plays an important role in the C2 cycle, is dicarboxylate antiport systems with overlapping substrate half-saturated at micromolar concentrations of oxaloacetate –1 specificities are involved in this process. The 2-oxoglu- (KmOAA = 5 µM; Vmax = 2 µmol min per mg of protein). tarate/malate translocator imports 2-oxoglutarate in The activity of this carrier appears to be high enough to exchange for stromal malate, whereas export of glutamate account for in vivo carbon fluxes through the inner mito- from the chloroplast in exchange for malate is catalysed by chondrial membrane. The purification and functional the glutamate/malate translocator. Malate is, therefore, the reconstitution, as well as the completion of detailed kinetic counterion for both translocators, resulting in 2-oxoglu- analyses, of this specific transporter should be undertaken. tarate/glutamate exchange without net malate import [47]. A cDNA clone encoding the spinach chloroplast 2-oxoglu- Porin of peroxisomal membrane tarate/malate translocator has been obtained by Weber et al. Apparently, peroxisomal membrane does not contain anion [48]. The predicted protein with an apparent molecular exchangers able to sustain the high fluxes of organic anions mass of 45 kDa contains a 12-helix motif and probably across the membrane of leaf peroxisomes. In fact, it has 220 Physiology and metabolism

been proposed that this membrane contains a slightly how the co-ordinated control of a multitude of genes in a anion-selective channel-forming component, in accordance precise spatial and temporal program, can lead to the with its physiological function and distinct from other development of this exquisite photorespiratory cycle. It known eukaryotic porins [51,52,53•]. For example, its sin- appears certain that the introduction of a genetic approach gle-channel conductance of about 300 pS (in 1 M KCl) is will complement the more classical methods used in the one order of magnitude lower than that of the mitochondr- study and regulation of photorespiration with regard to the ial porin. The narrow diameter (0.6 nm) of this ultimate goal of plants with superior growth pore-forming protein restricts the diffusion to anions (gly- characteristics and devising new herbicides. colate, glycerate, etc.). The characterization of a binding site for dicarboxylate anions inside the peroxisomal chan- Acknowledgements nel, however, is puzzling. It is possible, in analogy with This article is dedicated to the memory of Professor NE Tolbert, tireless inducible porins which have been characterized in some champion of photorespiration. gram-negative bacteria, that this binding site confers rather selective properties to this peroxisomal channel, preventing References and recommended reading the diffusion of highly reactive intermediates of peroxiso- Papers of particular interest, published within the annual period of review, • mal metabolism, such as glyoxylate and H2O2 [53 ]. have been highlighted as: • of special interest •• of outstanding interest Conclusions 1. Tolbert NE: The C2 oxidative photosynthetic . Annu It has been claimed that Rubisco behaves as a • Rev Plant Physiol Plant Mol Biol 1997, 48:1-25. ‘Schizophrenic’ enzyme because of its inability to protect This article provides an excellent overview of the C2 cycle. it’s ene-diolate reaction intermediate from O2 [13]. This 2. Husic DW, Husic HD, Tolbert NE: The oxidative photosynthetic • carbon cycle or C2 cycle. CRC Crit Rev Plant Sci 1987, unfair statement should be reconsidered [1 ], however, 5:45-100. because several groups have demonstrated that photorespi- 3. Leegood RC, Lea PJ, Adcok MD, Häusler RE: The regulation and ratory metabolism can prevent the formation of the excited control of photorespiration. J Exp Bot 1995, 46:1397-1414. triplet state of and excess reactive O2 species 4. Leegood RC, Lea PJ, Haüsler RE: Use of Barley mutants to study (superoxide radicals and singlet ) which necessarily the control of photorespiratory metabolism. Biochem Soc Trans 1996, 24:757-761. occur in sunlight when CO2, the final electron acceptor, is lacking [54]. In other words photorespiration, a very ‘waste- 5. Lea PJ, Ireland RJ: Nitrogen metabolism in higher plants. In The Plant Amino Acids. Edited by Singh BK. New York: Marcel Dekker ful’ process, in concert with other reactions including a Inc; 1998:1-47. cycle utilizing monodehydroascorbate reductase, dehy- 6. Marocco JP, Ku MSB, Lea PJ, Dever LV, Leegood RC, Furbank RT, droascorbate reductase and glutathione reductase • Edwards GE: Oxygen requirement and inhibition of C4 (Halliwell-Asada cycle) alleviates the damage that oxygen photosynthesis. Plant Physiol 1998, 116:823-832. This article clearly indicates that when the C4 cycle is ineffective in concen- radicals can cause in green leaves [55,56]. Wasteful and use- trating CO2 there is an increase in photorespiration. ful are not necessarily incompatibles and very likely 7. Von Caemmerer S, Evans JR, Hudson GS, Andrew TJ: The kinetics of Rubisco is more ‘clever’ than we thought because when ribulose-1,5-bisphosphate carboxylase/oxygenase in vivo inferred from measurements of photosynthesis in leaves of stomata are closed (the CO2 concentration of the intercel- transgenic tobacco. Planta 1994, 195:88-97. lular space of the leaves drops to the CO compensation 2 8. Bainbridge G, Madgwick P, Parmar S, Mitchell R, Paul M, Pitts J, Keys point) C3- and C2-cycles operate in perfect synchrony to AJ: Engineering Rubisco to change its catalytic properties. J Exp prevent excessive reduction, and, therefore, photoinactiva- Bot 1996, 46:1269-127. tion, of the chloroplast [55]. 9. Gutteridge S, Gatenby AA: Rubisco synthesis, assembly, mechanism and regulation. Plant Cell 1995, 7:809-819. Our understanding of which structural features of Rubisco 10. Cleland WW, Andrews TJ, Gutteridge S, Hartman FC, Lorimer, GH: •• Mechanism of Rubisco: the carbamate as general base. Chem control discrimination between the two gaseous substrates Rev 1998, 2:549-561. is rather meagre, and identification of determinants which A striking example of enzyme functioning. The divalent metal ion coordinat- ed lysyl carbamate plays the role of a cofactor and is central to our under- influence CO2 and O2 substrate specificities is a prerequi- standing of the carboxylation and oxygenation reactions. site for redirecting and modifying fluxes of glycolate-2-P 11. Wildner GF, Schlitter J, Müller M: Rubisco, an old challenge with and glycerate-3-P. Indeed, Rubisco is located at an ideal new perspectives. Z Naturforsch 1996, 51c:263-276. strategic position for control of photorespiration [8,11]. It is 12. Andersson I: Large structures at high resolution: the 1.6A crystal possible that the small subunit might influence both the structure of spinach Ribulose-1,5-bisphosphate carboxylase/oxygenase complexed with 2-carboxyarabinitol enzymatic turnover and the discrimination of the two bisphosphate. J Mol Biol 1996, 259:160-174. gaseous substrates [57,58•]. 13. Hartman FC, Harpel MR: Structure, function, regulation, and assembly of D-ribulose 1,5-bisphosphate Despite a few impressive advances, it is fair to say that we carboxylase/oxygenase. Annu Rev Biochem 1994, 63:197-234. still do not have a clear idea as to how any of these 14. Taylor TC, Andersson I: The structure of the complex between enzymes and transporters involved in photorespiratory •• Rubisco and its substrate ribulose 1,5-bisphosphate. J Mol Biol 1997, 265:432-444. cycle function at the molecular level in establishing the co- An impressive paper describing the mechanism of the enzyme in the light ordinated function of the C - C - and nitrogen-cycles for of the high-resolution structure. This paper gives the main structural fea- 3 2 tures of the active site which is situated at the carboxy-terminal end of the maximum efficiency. Likewise, an intriguing question is α/β-barrel of the L subunit. Biochemical dissection of photorespiration Douce and Neuburger 221

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