Crystal Structures of the Substrate-Bound Forms of Red Chlorophyll Catabolite Reductase: Implications for Site-Specific and Stereospecific Reaction

Red chlorophyll catabolite reductase (RCCR) catalyzes the ferredoxin-dependent reduction of the C20/C1 double bond of red chlorophyll catabolite (RCC), the catabolic intermediate produced in chlorophyll degradation. The crystal structure of substrate-free Arabidopsis thaliana RCCR (AtRCCR) demonstrated that RCCR folds into a characteristic α/β/α sandwich, similar to that observed in the ferredoxin-dependent bilin reductase (FDBR) family. Here we have determined the crystal structures of RCC-bound AtRCCR, RCC-bound F218V AtRCCR, and substrate-free F218V AtRCCR, a mutant protein that produces the stereoisomer of primary fluorescent chlorophyll catabolites at the C1 position. RCC is bound to the pocket between the β-sheet and the C-terminal α-helices, as seen in substrate-bound FDBRs, but RCC binding to RCCR is much looser than substrate binding to FDBRs. The loose binding seems beneficial to the large conformational change in RCC upon reduction. Two conserved acidic residues, Glu154 and Asp291, sandwich the C20/C1 double bond of RCC, suggesting that these two residues are involved in site-specific reduction. The RCC in F218V AtRCCR rotates slightly compared with that in wild type to fill in the space generated by the substitution of Phe218 with valine. Concomitantly, the two carboxy groups of Glu154 and Asp291 move slightly away from the C20/C1 double bond. The geometrical arrangement of RCC and the carboxy groups of Glu154 and Asp291 in RCCR would appear to be essential for the stereospecificity of the RCCR reaction.     

The chlorophyllases AtCLH1 and AtCLH2 are not essential for senescence-related chlorophyll breakdown in Arabidopsis thaliana

One important reaction of chlorophyll (chl) breakdown during plant senescence is the removal of the lipophilic phytol moiety by chlorophyllase. AtCLH1 and AtCLH2 were considered to be required for this reaction in Arabidopsis thaliana. Here we present evidence against this assumption. Using green fluorescent protein fusions, neither AtCLH isoform localizes to chloroplasts, the predicted site of chlorophyll breakdown. Furthermore, clh1 and clh2 single and double knockout lines are still able to degrade chlorophyll during senescence. From our data we conclude that AtCLHs are not required for senescence-related chlorophyll breakdown in vivo and propose that genuine chlorophyllase has not yet been molecularly identified.     

Evolution of Chlorophyll Degradation: The Significance of RCC Reductase

Abstract: In angiosperms the key process of chlorophyll breakdown in senescing leaves is catalyzed by pheophorbide a oxygenase and RCC reductase which, in a metabolically channeled reaction, cleave the porphyrin macrocycle and produce a colourless primary catabolite, pFCC. RCC reductase is responsible for the reduction of the C20/C1 double bond of the intermediary catabolite, RCC. Depending on plant species, RCC reductase produces one of the two C1 stereoisomers, pFCC-1 or pFCC-2. Screening of a large number of taxa for the type of RCCR revealed that the isomer produced is uniform within families. It also revealed that type RCCR-2 is predominant; RCCR-1 seems to represent a recent derivation which in unrelated lineages has evolved independently from RCCR-2. A third type of pFCC was produced by RCCR from basal pteridophytes and some gymnosperms; its structure is unknown. Collectively, the data suggest that the pathway of chlorophyll breakdown is very conserved in vascular plants. RCCR appears to represent a decisive addition to the catabolic pathway: it allows terrestrial plants to metabolize the porphyrin part of the chlorophyll molecule to photodynamically inactive final products that are stored in the vacuoles of senescing mesophyll cells.     

Correlation of leaf senescence and gene expression/activities of chlorophyll degradation enzymes in harvested Chinese flowering cabbage (Brassica rapa var. parachinensis)

Chinese flowering cabbage is one of the main leafy vegetables produced in China. They have a rapid leaf yellowing due to chlorophyll degradation after harvest that limits their marketing. In the present study, leaf senescence of the cabbages was manipulated by ethylene and 6-benzyl aminopurine (6-BA) treatment to investigate the correlation of leaf senescence and chlorophyll degradation related to gene expression/activities in the darkness. The patterns of several senescence associated markers, including a typical marker, the expression of senescence-associated gene SAG₁₂, demonstrated that ethylene accelerated leaf senescence of the cabbages, while 6-BA retarded this progress. Similar to the trends of BrSAG₁₂ gene expression, strong activation in the expression of three chlorophyll degradation related genes, pheophytinase (BrPPH), pheophorbide a oxygenase (BrPAO) and red chlorophyll catabolite reductase (BrRCCR), was detected in ethylene treated and control leaves during the incubation, while no evident increase was recorded in 6-BA treated leaves. The overall dynamics of Mg-dechelatase activities in all treatments displayed increasing trends during the senescence process, and a delayed increase in the activities was observed for 6-BA treated leaves. However, chlorophyllase activity as well as the expression of BrChlase1 and BrChlase2 decreased with the incubation in all treatments. Taken together, the expression of BrPPH, BrPAO and BrRCCR, and the activity of Mg-dechelatase was closely associated with the chlorophyll degradation during the leaf senescence process in harvested Chinese flowering cabbages under dark conditions.     

Glutathione Reductase and Thioredoxin Reductase: Novel Antioxidant Enzymes from Plasmodium berghei

Malaria parasites adapt to the oxidative stress during their erythrocytic stages with the help of vital thioredoxin redox system and glutathione redox system. Glutathione reductase and thioredoxin reductase are important enzymes of these redox systems that help parasites to maintain an adequate intracellular redox environment. In the present study, activities of glutathione reductase and thioredoxin reductase were investigated in normal and Plasmodium berghei-infected mice red blood cells and their fractions. Activities of glutathione reductase and thioredoxin reductase in P. berghei-infected host erythrocytes were found to be higher than those in normal host cells. These enzymes were mainly confined to the cytosolic part of cell-free P. berghei. Full characterization and understanding of these enzymes may promise advances in chemotherapy of malaria.     

In vivo participation of red chlorophyll catabolite reductase in chlorophyll breakdown

A central reaction of chlorophyll breakdown, porphyrin ring opening of pheophorbide a to the primary fluorescent chlorophyll catabolite (pFCC), requires pheophorbide a oxygenase (PAO) and red chlorophyll catabolite reductase (RCCR), with red chlorophyll catabolite (RCC) as a presumably PAO-bound intermediate. In subsequent steps, pFCC is converted to different fluorescent chlorophyll catabolites (FCCs) and nonfluorescent chlorophyll catabolites (NCCs). Here, we show that RCCR-deficient Arabidopsis thaliana accumulates RCC and three RCC-like pigments during senescence, as well as FCCs and NCCs. We also show that the stereospecificity of Arabidopsis RCCR is defined by a small protein domain and can be reversed by a single Phe-to-Val exchange. Exploiting this feature, we prove the in vivo participation of RCCR in chlorophyll breakdown. After complementation of RCCR mutants with RCCRs exhibiting alternative specificities, patterns of chlorophyll catabolites followed the specificity of complementing RCCRs. Light-dependent leaf cell death observed in different RCCR-deficient lines strictly correlated with the accumulation of RCCs and the release of singlet oxygen, and PAO induction preceded lesion formation. These findings suggest that RCCR absence causes leaf cell death as a result of the accumulation of photodynamic RCC. We conclude that RCCR (together with PAO) is required for the detoxification of chlorophyll catabolites and discuss the biochemical role(s) for this enzyme.     

Chlorophyll breakdown in oilseed rape

Chlorophyll catabolism accompanying leaf senescence is one of the most spectacular natural phenomena. Despite this fact, the metabolism of chlorophyll has been largely neglegted until recently. Oilseed rape has been used extensively as a model plant for the recent elucidating of structures of chlorophyll catabolites and for investigation of the enzymic reactions of the chlorophyll breakdown pathway. The key reaction which causes loss of green color is catalyzed in a two-step reaction by pheophorbide a oxygenase and red chlorophyll catabolite reductase. In this Minireview, we summarize the actual knowledge about catabolites and enzymes of chlorophyll catabolism in oilseed rape and discuss the significance of this pathway in respect to chlorophyll degradation during Brassica napus seed development. This revised version was published online in June 2006 with corrections to the Cover Date.     

Phycoerythrobilin synthase (PebS) of a marine virus. Crystal structures of the biliverdin complex and the substrate-free form

The reddish purple open chain tetrapyrrole pigment phycoerythrobilin (PEB; A(lambdamax) approximately 550 nm) is an essential chromophore of the light-harvesting phycobiliproteins of most cyanobacteria, red algae, and cryptomonads. The enzyme phycoerythrobilin synthase (PebS), recently discovered in a marine virus infecting oceanic cyanobacteria of the genus Prochlorococcus (cyanophage PSSM-2), is a new member of the ferredoxin-dependent bilin reductase (FDBR) family. In a formal four-electron reduction, the substrate biliverdin IXalpha is reduced to yield 3Z-PEB, a reaction that commonly requires the action of two individual FDBRs. The first reaction catalyzed by PebS is the reduction of the 15,16-methine bridge of the biliverdin IXalpha tetrapyrrole system. This reaction is exclusive to PEB biosynthetic enzymes. The second reduction site is the A-ring 2,3,3(1),3(2)-diene system, the most common target of FDBRs. Here, we present the first crystal structures of a PEB biosynthetic enzyme. Structures of the substrate complex were solved at 1.8- and 2.1-A resolution and of the substrate-free form at 1.55-A resolution. The overall folding revealed an alpha/beta/alpha-sandwich with similarity to the structure of phycocyanobilin:ferredoxin oxidoreductase (PcyA). The substrate-binding site is located between the central beta-sheet and C-terminal alpha-helices. Eight refined molecules with bound substrate, from two different crystal forms, revealed a high flexibility of the substrate-binding pocket. The substrate was found to be either in a planar porphyrin-like conformation or in a helical conformation and is coordinated by a conserved aspartate/asparagine pair from the beta-sheet side. From the alpha-helix side, a conserved highly flexible aspartate/proline pair is involved in substrate binding and presumably catalysis.     

Inactivation of the geranylgeranyl reductase (ChlP) gene in the cyanobacterium Synechocystis sp. PCC 6803

Geranylgeranyl reductase catalyses the reduction of geranylgeranyl pyrophosphate to phytyl pyrophosphate required for synthesis of chlorophylls, phylloquinone and tocopherols. The gene chlP (ORF sll1091) encoding the enzyme has been inactivated in the cyanobacterium Synechocystis sp. PCC 6803. The resulting ΔchlP mutant accumulates exclusively geranylgeranylated chlorophyll a instead of its phytylated analogue as well as low amounts of α-tocotrienol instead of α-tocopherol. Whereas the contents of chlorophyll and total carotenoids are decreased, abundance of phycobilisomes is increased in ΔchlP cells. The mutant assembles functional photosystems I and II as judged from 77 K fluorescence and electron transport measurements. However, the mutant is unable to grow photoautotrophically due to instability and rapid degradation of the photosystems in the absence of added glucose. We suggest that instability of the photosystems in ΔchlP is directly related to accumulation of geranylgeranylated chlorophyll a. Increased rigidity of the chlorophyll isoprenoid tail moiety due to three additional CC bonds is the likely cause of photooxidative stress and reduced stability of photosynthetic pigment-protein complexes assembled with geranylgeranylated chlorophyll a in the ΔchlP mutant.     

Crystal Structure and Catalytic Mechanism of Leucoanthocyanidin Reductase from Vitis vinifera

Leucoanthocyanidin reductase (LAR) catalyzes the NADPH-dependent reduction of 2R,3S,4S-flavan-3,4-diols into 2R,3S-flavan-3-ols, a subfamily of flavonoids that is important for plant survival and for human nutrition. LAR1 from Vitis vinifera has been co-crystallized with or without NADPH and one of its natural products, (+)-catechin. Crystals diffract to a resolution between 1.75 and 2.72 Å. The coenzyme and substrate binding pocket is preformed in the apoprotein and not markedly altered upon NADPH binding. The structure of the abortive ternary complex, determined at a resolution of 2.28 Å, indicates the ordering of a short 3₁₀ helix associated with substrate binding and suggests that His122 and Lys140 act as acid-base catalysts. Based on our 3D structures, a two-step catalytic mechanism is proposed, in which a concerted dehydration precedes an NADPH-mediated hydride transfer at C4. The dehydration step involves a Lys-catalyzed deprotonation of the phenolic OH7 through a bridging water molecule and a His-catalyzed protonation of the benzylic hydroxyl at C4. The resulting quinone methide serves as an electrophilic target for hydride transfer at C4. LAR belongs to the short-chain dehydrogenase/reductase superfamily and to the PIP (pinoresinol-lariciresinol reductase, isoflavone reductase, and phenylcoumaran benzylic ether reductase) family. Our data support the concept that all PIP enzymes reduce a quinone methide intermediate and that the major role of the only residue that has been conserved from the short-chain dehydrogenase/reductase catalytic triad (Ser…TyrXXXLys), that is, lysine, is to promote the formation of this intermediate by catalyzing the deprotonation of a phenolic hydroxyl. For some PIP enzymes, this lysine-catalyzed proton abstraction may be sufficient to trigger the extrusion of the leaving group, whereas in LAR, the extrusion of a hydroxide group requires a more sophisticated mechanism of concerted acid-base catalysis that involves histidine and takes advantage of the OH4, OH5, and OH7 substituents of leucoanthocyanidins.     

Insights into phycoerythrobilin biosynthesis point toward metabolic channeling

Phycoerythrobilin is a linear tetrapyrrole molecule found in cyanobacteria, red algae, and cryptomonads. Together with other bilins such as phycocyanobilin it serves as a light-harvesting pigment in the photosynthetic light-harvesting structures of cyanobacteria called phycobilisomes. The biosynthesis of both pigments starts with the cleavage of heme by heme oxygenases to yield biliverdin IXalpha, which is further reduced at specific positions by ferredoxin-dependent bilin reductases (FDBRs), a new family of radical enzymes. The biosynthesis of phycoerythrobilin requires two subsequent two-electron reductions, each step being catalyzed by one FDBR. This is in contrast to the biosynthesis of phycocyanobilin, where the FDBR phycocyanobilin: ferredoxin oxidoreductase (PcyA) catalyzes a four-electron reduction. The first reaction in phycoerythrobilin biosynthesis is the reduction of the 15,16-double bond of biliverdin IXalpha by 15,16-dihydrobiliverdin:ferredoxin oxidoreductase (PebA). This reaction reduces the conjugated pi -electron system thereby blue-shifting the absorbance properties of the linear tetrapyrrole. The second FDBR, phycoerythrobilin:ferredoxin oxidoreductase (PebB), then reduces the A-ring 2,3,3(1),3(2)-diene structure of 15,16-dihydrobiliverdin to yield phycoerythrobilin. Both FDBRs from the limnic filamentous cyanobacterium Fremyella diplosiphon and the marine cyanobacterium Synechococcus sp. WH8020 were recombinantly produced in Escherichia coli and purified, and their enzymatic activities were determined. By using various natural bilins, the substrate specificity of each FDBR was established, revealing conformational preconditions for their unique specificity. Preparation of the semi-reduced intermediate, 15,16-dihydrobiliverdin, enabled us to perform steady state binding experiments indicating distinct spectroscopic and fluorescent properties of enzyme.bilin complexes. A combination of substrate/product binding analyses and gel permeation chromatography revealed evidence for metabolic channeling.     

Mild ammonium stress increases chlorophyll content in Arabidopsis thaliana

Nitrate (NO₃⁻) and ammonium (NH₄⁺) are the main forms of nitrogen available in the soil for plants. Excessive NH₄⁺ accumulation in tissues is toxic for plants and exclusive NH₄⁺-based nutrition enhances this effect. Ammonium toxicity syndrome commonly includes growth impairment, ion imbalance and chlorosis among others. In this work, we observed high intraspecific variability in chlorophyll content in 47 Arabidopsis thaliana natural accessions grown under 1 mM NH₄⁺ or 1 mM NO₃⁻ as N-source. Interestingly, chlorophyll content increased in every accession upon ammonium nutrition. Moreover, this increase was independent of ammonium tolerance capacity. Thus, chlorosis seems to be an exclusive effect of severe ammonium toxicity while mild ammonium stress induces chlorophyll accumulation.     

Structure and mutation analysis of archaeal geranylgeranyl reductase

The crystal structure of geranylgeranyl reductase (GGR) from Sulfolobus acidocaldarius was determined in order to elucidate the molecular mechanism of the catalytic reaction. The enzyme is a flavoprotein and is involved in saturation of the double bonds on the isoprenoid moiety of archaeal membranes. The structure determined in this study belongs to the p-hydroxybenzoate hydroxylase family in the glutathione reductase superfamily. GGR functions as a monomer and is divided into the FAD-binding, catalytic and C-terminal domains. The catalytic domain has a large cavity surrounded by a characteristic YxWxFPx_7-8GxG motif and by the isoalloxazine ring of an FAD molecule. The cavity holds a lipid molecule, which is probably derived from Escherichia coli cells used for over-expression. One of the two forms of the structure clarifies the presence of an anion pocket holding a pyrophosphate molecule, which might anchor the phosphate head of the natural ligands. Mutational analysis supports the suggestion that the three aromatic residues of the YxWxFPx_7-8GxG motif hold the ligand in the appropriate position for reduction. Cys47, which is widely conserved in GGRs, is located at the si-side of the isoalloxazine ring of FAD and is shown by mutational analysis to be involved in catalysis. The catalytic cycle, including the FAD reducing factor binding site, is proposed on the basis of the detailed analysis of the structure.     

Participation of Chlorophyll b Reductase in the Initial Step of the Degradation of Light-harvesting Chlorophyll a/b-Protein Complexes in Arabidopsis

The light-harvesting chlorophyll a/b-protein complex of photosystem II (LHCII) is the most abundant membrane protein in green plants, and its degradation is a crucial process for the acclimation to high light conditions and for the recovery of nitrogen (N) and carbon (C) during senescence. However, the molecular mechanism of LHCII degradation is largely unknown. Here, we report that chlorophyll b reductase, which catalyzes the first step of chlorophyll b degradation, plays a central role in LHCII degradation. When the genes for chlorophyll b reductases NOL and NYC1 were disrupted in Arabidopsis thaliana, chlorophyll b and LHCII were not degraded during senescence, whereas other pigment complexes completely disappeared. When purified trimeric LHCII was incubated with recombinant chlorophyll b reductase (NOL), expressed in Escherichia coli, the chlorophyll b in LHCII was converted to 7-hydroxymethyl chlorophyll a. Accompanying this conversion, chlorophylls were released from LHCII apoproteins until all the chlorophyll molecules in LHCII dissociated from the complexes. Chlorophyll-depleted LHCII apoproteins did not dissociate into monomeric forms but remained in the trimeric form. Based on these results, we propose the novel hypothesis that chlorophyll b reductase catalyzes the initial step of LHCII degradation, and that trimeric LHCII is a substrate of LHCII degradation.     

Plant degreening: evolution and expression of tomato (Solanum lycopersicum) dephytylation enzymes

Chlorophyll is the most abundant pigment on earth and even though it is known that its high photo-excitability necessitates a tight regulation of its degradation pathway, to date there are still several steps in chlorophyll breakdown that remain obscure. In order to better understand the ‘degreening’ processes that accompany leaf senescence and fruit ripening, we characterized the enzyme-encoding genes involved in dephytylation from tomato (Solanum lycopersicum). A single pheophytinase (PPH) gene and four chlorophyllase (CLH) genes were identified in the tomato genome. A phenetic analysis revealed two groups of CLHs in eudicot species and further evolutionary analysis indicated that these enzymes are under diverse selection pressures. A comprehensive expression profile analysis also suggested functional specificity for these dephytylating enzymes. The integrated analysis allows us to propose three general roles for chlorophyll dephytylation: i) PPH, which is under high selective constraint, is responsible for chlorophyll degradation during developmentally programed physiological processes; ii) Group I CLHs, which are under relaxed selection constraint, respond to environmental and hormonal stimuli and play a role in plant adaptation plasticity; and iii) Group II CLHs, which are also under high selective constraint, are mostly involved in chlorophyll recycling.     

Phytochemical analysis of Jatropha curcas L. during different seasons and developmental stages and seedling growth of wheat (Triticum aestivum L) as affected by extracts/leachates of Jatropha curcas L

Jatropha curcas shows invasive characters and is a significant source of many phytochemicals with varying biological activities. Different plant parts of Jatropha curcas L exhibited variation in their phytochemical constituents. Leaves and ovary walls were found to contain higher contents of total phenols, tannins and phytic acid whereas free amino acids were greater in leaves. Young leaves of Jatropha show greater contents of all these metabolites. Further, plants exhibit seasonal differences as leaves collected during summer (May-June) have greater accumulation of total phenols, tannins and free amino acids however, phytic acid was more during rainy season. Leachates and extracts in their higher concentrations adversely affected the germination and growth of wheat seedlings however, lower concentrations were more or less stimulatory. These treatments not only decreased the length, fresh and dry weight of seedlings but also affected the chlorophyll contents and activity of enzymes such as nitrate reductase, aminotransferases in wheat seedlings however, the activity of superoxide dismutase and ascorbate peroxidases increased. Experiments indicate harmful allelopathic effects of Jatropha leachates /extracts on wheat seedlings, hence further experimentation and analysis is recommended before continued plantation of Jatropha particularly on fertile soils. However. Growth of Jatropha plants on saline soils and their potential for accumulating sodium, potassium and chloride are the attributes suggesting the possibility of use of Jatropha plants in improving saline soils.