Evidence for the Presence of Chlorophyllide b in the Green Alga Scenedesmus obliquus in vivo

Chlorophyllide b could be extracted from the wild type of Scenedesmus obliquus and its pigment mutant C-2A'. Its identity was proved by absorption and fluorescence spectroscopy and by a positive hydroxylamine test. Chlorophyllide b could be transformed into pheophorbide b and methylpheophorbide b. The formation of chlorophyllide b from chlorophyll b by dephytylation with chlorophyllase could be ruled out. The stimulation of chlorophyllide b biosynthesis with o-phenanthroline, as described in the literature, could not be confirmed under physiological conditions.     

Pre-loading of chlorophyll synthase with tetraprenyl diphosphate is an obligatory step in chlorophyll biosynthesis

The reaction of recombinant chlorophyll synthase from Avena sativa, expressed in Escherichia coli, was investigated. To verify the identity of the recombinant and native enzymes, reaction rates were determined for both enzyme preparations with several chlorophyllide analogs. The rates of esterification of these modified substrates ranged from 0 to 100% of the rate with the natural substrate, and were nearly identical for both enzyme preparations. The Lineweaver-Burk plot for variation of both chlorophyllide a and phytyl diphosphate concentration showed parallel lines, indicative of a 'ping-pong' mechanism. Pre-incubation with phytyl diphosphate exhibited an initial rapid reaction phase, which did not occur after pre-incubation with chlorophyllide. We conclude that the tetraprenyl diphosphate must bind to the enzyme as the first substrate and esterification occurs when this pre-loaded enzyme meets the second substrate, chlorophyllide. Approximately 10-17% of the recombinant enzyme were pre-loaded with phytyl diphosphate under the experimental conditions. The rapid reaction phase is also observed for the chlorophyll synthase reaction in etiolated barley leaves in addition to the well-known slow phase. This indicates that pre-loading of the enzyme with tetraprenyl diphosphate is also the basis for the reaction in vivo.     

Identification of the 7-hydroxymethyl chlorophyll a reductase of the chlorophyll cycle in Arabidopsis

The interconversion of chlorophyll a and chlorophyll b, referred to as the chlorophyll cycle, plays a crucial role in the processes of greening, acclimation to light intensity, and senescence. The chlorophyll cycle consists of three reactions: the conversions of chlorophyll a to chlorophyll b by chlorophyllide a oxygenase, chlorophyll b to 7-hydroxymethyl chlorophyll a by chlorophyll b reductase, and 7-hydroxymethyl chlorophyll a to chlorophyll a by 7-hydroxymethyl chlorophyll a reductase. We identified 7-hydroxymethyl chlorophyll a reductase, which is the last remaining unidentified enzyme of the chlorophyll cycle, from Arabidopsis thaliana by genetic and biochemical methods. Recombinant 7-hydroxymethyl chlorophyll a reductase converted 7-hydroxymethyl chlorophyll a to chlorophyll a using ferredoxin. Both sequence and biochemical analyses showed that 7-hydroxymethyl chlorophyll a reductase contains flavin adenine dinucleotide and an iron-sulfur center. In addition, a phylogenetic analysis elucidated the evolution of 7-hydroxymethyl chlorophyll a reductase from divinyl chlorophyllide vinyl reductase. A mutant lacking 7-hydroxymethyl chlorophyll a reductase was found to accumulate 7-hydroxymethyl chlorophyll a and pheophorbide a. Furthermore, this accumulation of pheophorbide a in the mutant was rescued by the inactivation of the chlorophyll b reductase gene. The downregulation of pheophorbide a oxygenase activity is discussed in relation to 7-hydroxymethyl chlorophyll a accumulation.     

Overexpression of chlorophyllide a oxygenase (CAO) enlarges the antenna size of photosystem II in Arabidopsis thaliana

The light-harvesting efficiency of a photosystem is thought to be largely dependent on its photosynthetic antenna size. It has been suggested that antenna size is controlled by the biosynthesis of chlorophyll b. To verify this hypothesis, we overexpressed the enzyme for chlorophyll b biosynthesis, chlorophyllide a oxygenase (CAO), in Arabidopsis thaliana by transforming the plant with cDNA for CAO under the control of the 35S cauliflower mosaic virus promoter. In the early de-etiolation phase, when the intrinsic CAO expression is very low, the chlorophyll a: b ratio was drastically decreased from 28 to 7.3, indicating that enhancement of chlorophyll b biosynthesis had been successfully achieved. We made the following observations in full-green rosette leaves of transgenic plants. (1) The chlorophyll a : b ratio was reduced from 2.85 to 2.65. (2) The ratio of the peripheral light-harvesting complexes (LHCII) to the core antenna complex (CPa) resolved with the green-gel system increased by 20%. (3) The ratio of the light-harvesting complex II apoproteins (LHCP) to 47-kDa chlorophyll a protein (CP47), which was estimated by the results of immunoblotting, increased by 40%. These results indicated that the antenna size increased by at least 10-20% in transgenic plants, suggesting that chlorophyll b biosynthesis controls antenna size. To the best of our knowledge, this is the first report on enlargement of the antenna size by genetic manipulations.     

Chlorophyll a and b biosynthesis in the dark in etiolated leaves infiltrated by exogenous chlorophyllide a]

Exogenous chlorophyllide a was introduced into etiolated rye leaves by the vacuum-infiltration technique. Appearance and accumulation of chlorophylls a and b within the leaves are observed during continued darkening, protochlorophyllide photoreduction being avoided. The pigments are identified by the solubility in petroleum ether, paper chromatograms, the fluorescence maxima, the peculiarities of exciting light 430 and 460 nm effects on fluorescence intensity, the specific interaction with hydrochloric hydroxylamine. The conclusion is made that before illumination etioplasts already contain enzyme systems and substrates which provide esterification of chlorophyllide a to chlorophyll a and conversion of chlorophyll a into chlorophyll b.     

Chloroplast biogenesis: [4-vinyl] chlorophyllide a reductase is a divinyl chlorophyllide a-specific, NADPH-dependent enzyme.

Some properties of [4-vinyl] chlorophyllide a reductase are described. This enzyme converts divinyl chlorophyllide a to monovinyl chlorophyllide a. The latter is the immediate precursor of monovinyl chlorophyll a, the main chlorophyll in green plants. [4-Vinyl] chlorophyllide a reductase plays an important role in daylight during the conversion of divinyl protochlorophyllide a to monovinyl chlorophyll a. [4-Vinyl] chlorophyllide a reductase was detected in isolated plastid membranes. Its activity is strictly dependent on the availability of NADPH. Other reductants such as NADH and GSH were ineffective. The enzyme appears to be specific for divinyl chlorophyllide a, and it does not reduce divinyl protochlorophyllide a to monovinyl protochlorophyllide a. The conversion of divinyl protochlorophyllide a to monovinyl protochlorophyllide a has been demonstrated in barley and cucumber etiochloroplasts and appears to be catalyzed by a [4-vinyl] protochlorophyllide a reductase [Tripathy, B.C., & Rebeiz, C.A. (1988) Plant Physiol. 87, 89-94]. On the basis of reductant requirements and substrate specificity, it is possible that two different 4-vinyl reductases may be involved in the reduction of divinyl protochlorophyllide a and divinyl chlorophyllide a to their respective 4-ethyl analogues.     

Esterification of chlorophyllide b in higher plants

Chlorophyllide b and four chemically different chlorophyll b specieis, chlorophyllide b esterified with geranylgeraniol, dihydrogeranylgeraniol, tetrahydrogeranylgeraniol and phytol have been detected in addition to the same derivatives of chlorophyll a in the greening cotyledons of cucumber. These esters could be separated and determined by high-performance liquid chromatography. The results suggest that chlorophyll b phytol is formed from the esterification of chlorophyllide b and geranylgeraniol followed by three hydrogenations of the alcohol moiety, as in the case of chlorophyll a and protochlorophyll phytol formation     

Chlorophyll b inhibits the formation of photosystem I trimer in Synechocystis sp. PCC6803

Chlorophyllide a oxygenase (CAO) catalyzes two-step oxygenation reactions and converts chlorophyllide a to chlorophyllide b. When CAO was introduced into the Synechocystis sp. PCC6803 genome, chlorophyll b was synthesized and incorporated into P700-chlorophyll a-protein complexes. Curve analysis of photosystem I particles showed that Ca687 was decreased with a concomitant increase in Cb652 suggesting that chlorophyll b was incorporated into Ca687-binding sites. When the level of chlorophyll b was high, Ca704, which is known as red chlorophyll and photosystem I trimers were decreased. Formation of photosystem I trimers is discussed in relation to red chlorophyll and chlorophyll b accumulation.     

Inhibition of chlorophyll biosynthesis by α′,α′-dipyridyl during greening of groundnut leaves

The site of inhibition of chlorophyll biosynthesis by α′,α′-dipyridyl was found to be at the level of conversion of chlorophyllide (672 nm) to chlorophyll (678 nm) during greening of groundnut leaves. This inhibition was partially reversed by certain divalent cations.     

Pigment shuffling in antenna systems achieved by expressing prokaryotic chlorophyllide a oxygenase in Arabidopsis

The organization of pigment molecules in photosystems is strictly determined. The peripheral antennae have both chlorophyll a and b, but the core antennae consist of only chlorophyll a in green plants. Furthermore, according to the recent model obtained from the crystal structure of light-harvesting chlorophyll a/b-protein complexes II (LHCII), individual chlorophyll-binding sites are occupied by either chlorophyll a or chlorophyll b. In this study, we succeeded in altering these pigment organizations by introducing a prokaryotic chlorophyll b synthesis gene (chlorophyllide a oxygenase (CAO)) into Arabidopsis. In these transgenic plants (Prochlirothrix hollandica CAO plants), approximately 40% of chlorophyll a of the core antenna complexes was replaced by chlorophyll b in both photosystems. Chlorophyll a/b ratios of LHCII also decreased from 1.3 to 0.8 in PhCAO plants. Surprisingly, these transgenic plants were capable of photosynthetic growth similar to wild type under low light conditions. These results indicate that chlorophyll organizations are not solely determined by the binding affinities, but they are also controlled by CAO. These data also suggest that strict organizations of chlorophyll molecules are not essential for photosynthesis under low light conditions.     

Studies on chlorophyllase of Chlorella protothecoides III. Purification and catalytic properties

Chlorophyllase was extracted from green cells of Chlorella protothecoidesby n-butanol treatment and purified 600-fold, as measured byenzyme activity in chlorophyll a hydrolysis, by ammonium sulfateprecipitation, chromatography on TEAE-cellulose column and gelfiltration with Sephadex G-200. At each purification step the following activities were compared:hydrolyses of chlorophyll a and methyl chlorophyllide a, methanolysisof chlorophyll a and transphythylation of methyl chlorophyllidea to chlorophyll a. The ratio of activities of chlorophyll a hydrolysis to chlorophylla methanolysis changed on purification and partial inactivationby heat, PCMB and phytol, as well as by varying the reactiontemperature, thus suggesting that the two reactions are notcatalyzed by a single enzyme. In contrast, the activity ratio of chlorophyll a methanolysisto transphytylation of methyl chlorophyllide a remained unaltered,indicating that these reactions can be forward and backwardreactions catalyzed by one enzyme. Results of kinetic studies also indicated that the chlorophyllaseof Chlorella protothecoides consists of at least two enzymes.One enzyme catalyzes chlorophyll a hydrolysis and the other,chlorophyll a methanolysis and the reverse reaction, transphytylationof methyl chlorophyllide a. (Received May 24, 1973; )     

Cloning and functional expression of the gene encoding the key enzyme for chlorophyll b biosynthesis (CAO) from Arabidopsis thaliana

Chlorophyll (Chl) biosynthesis and degradation are the only biochemical processes on Earth that can be directly observed from satellites or other planets. The bulk of the Chls is found in the light-harvesting antenna complexes of photosynthetic organisms. Surprisingly little is known about the biosynthesis of Chl b, which is the second most abundant Chl pigment after Chl a. We describe here the expression and properties of the chlorophyllide a oxygenase gene (CAO) from Arabidopsis thaliana, which is apparently the key enzyme in Chl b biosynthesis. The recombinant enzyme produced in Escherichia coli catalyses an unusual two-step oxygenase reaction that is the 'missing link' in the chlorophyll cycle of higher plants.     

Localization of chlorophyllase in the chloroplast envelope

Chlorophyllase catalyzes the first step in the catabolic pathway of chlorophyll. It is a constitutive enzyme located in chloroplast membranes. In isolated plastids the hydrolysis of the endogenous chlorophyll does not take place unless the membranes are solubilized in the presence of detergent. The structural latency of chlorophyllase activity appears to be due to the differential locations of substrate and enzyme within the plastids. Envelope membranes prepared from both chloroplasts and gerontoplasts contain chlorophyllase activity. The isolation of envelopes is associated with a marked increase in chlorophyllase activity per unit of protein. Yields of chlorophyllase and of specific envelope markers in the final preparations are similar, suggesting that the enzyme may be located in the envelope. It is hypothesized that the breakdown of chlorophyll during leaf senescence requires a mechanism that mediates the transfer of chlorophyll from the thylakoidal pigment-protein complexes to the sites of catabolic reactions in the envelope.Abbreviations ACT acyl CoA thioesterase - Chl chlorophyll - Chlide chlorophyllide - PC phosphatidylcholine     

Enzymes of the last steps of chlorophyll biosynthesis: modification of the substrate structure helps to understand the topology of the active centers

Enzymes catalyzing two of the late steps of chlorophyll biosynthesis are NADPH:protochlorophyllide oxidoreductase (POR), responsible for the light-dependent reduction of protochlorophyllide to chlorophyllide, and chlorophyll synthase that catalyses the esterification of chlorophyllide to chlorophyll. Inhibitors of these enzymes are of interest as potential herbicides. Both enzymes presumably form a complex, and the question arose whether chlorophyll synthase can react with chlorophyllide while it is still bound to POR. Here, we describe the chemical modification of protochlorophyllides and chlorophyllides with space-filling substituents at rings A, B, and E of the tetrapyrrole macrocycle and the reactivity of the modified substrates. Both enzymes tolerate the large and flexible phenylamino substituent at ring B, indicating that ring B points toward the enzyme surface while the substrate is bound. On the basis of the standard compound zinc protopheophorbide a (100% activity), the 7(1)-phenylamino derivative shows a comparable activity (83%) with POR that is higher than that of the parent formyl derivative zinc protopheophorbide b (58% activity). In contrast, the 3(1)-phenylamino derivative is less active (12%) than the parent formyl compound zinc protopheophorbide d (49% activity), indicating that the binding pocket leaves less space around ring A than around ring B. Almost no space must be left around ring E because substitution of the 13(2)-carboxymethyl ester (100% activity) by the 13(2)-carboxyethyl ester reduces the activity to 0.2%. Chlorophyll synthase leaves somewhat more space around ring E on the A side of the tetrapyrrole in the binding pocket; substitution of the 13(2)-proton (100% activity) by a methoxy group (53% activity) and an ethoxy group (11% activity) is tolerated to a certain extent, while the carbomethoxy group in this position is not accepted. Opening of ring E to a chlorin e6 dimethylester is tolerated (39% activity), while the large benzylamide residue at this site leads to the loss of activity. We conclude that the tetrapyrroles bind to both enzymes in the same direction: rings C, D, and E are oriented to the interior of the binding cleft, and rings A and B are oriented to the surface of the enzyme; this excludes simultaneous binding to both enzymes.     

Chlorophyll synthetase is latent in well preserved prolamellar bodies of etiolated wheat

Membrane fractions containing intact etioplasts, etioplast inner membranes, prolamellar bodies or prothylakoids from wheat ( Triticum aestivum L. cv. Walde) were assayed for chlorophyll synthetase activity. Calculated on a protein basis, the etioplast inner membrane fraction showed a higher activity than the intact etioplasts. The activity was higher in the prolamellar body fraction than in the prothylakoid fraction. However, when the fractions were incubated in isolation medium with 50% (w/w) sucrose and 0.3 m M NADPH, chlorophyll synthetase activity could not be detected in the prolamellar body fraction, while the prothylakoid fraction maintained a high activity. The spectral shift to a shorter wavelength of the newly formed endogenous chlorophyllide was very rapid in the prothylakoid fraction but slow in the prolamellar body fraction. The relation between the spectral shift of chlorophyllide and the esterification activity in the fractions is discussed. Even exogenous short-wavelength chlorophyllide could not be esterified in well preserved prolamellar bodies. This indicates that chlorophyll synthetase is present in an inactive state in the prolamellar body structure. A large-scale method for the synthesis of geranylgeranylpyrophosphate, one of the substrates of the chlorophyll synthetase reaction, is also presented.     

Analysis of the chlorophyll biosynthetic system in a chlorophyll b-less barley mutant

The chlorophyll b-less barley (Hordeum vulgare L.) mutant chlorina 2807 allelic to the well-known barley mutant chlorina f2 was studied. 5-Aminolevulinic acid at saturating concentration (40 mM) was introduced into postetiolated leaves of the mutant and its wild type, and the protochlorophyllide accumulation in the dark was measured. It was found that the activity of the enzyme system transforming 5-aminolevulinic acid into protochlorophyllide was the same in both types of plants. The activity of esterifying enzymes that catalyze attachment of phytol to chlorophyllide was analyzed by infiltration of exogenous chlorophyllides a and b into etiolated leaves. The reaction was shown to have close rates in the mutant and wild-type plants. In very early stages of greening of etiolated leaves, when the apoproteins of the light-harvesting complexes are not yet formed, appearance of chlorophyll b was clearly recorded in the wild-type plants, while in the mutant chlorina 2807 no indications of chlorophyll b were detected in any stage of greening. On the other hand, in the mutant as well as in the wild type an active reverse conversion of chlorophyll b into chlorophyll a was possible. It is concluded that (a) in the mutant chlorina 2807 the ability of the biosynthetic system to transform 5-aminolevulinic acid to chlorophyll a is fully preserved, (b) in the mutant the enzymes converting chlorophyll a into chlorophyll b are most likely absent or damaged, (c) the conversion of chlorophyll a into chlorophyll b and the reverse conversion of chlorophyll b into chlorophyll a are performed by different enzymes.     

Chlorophyll degradation in a Chlamydomonas reinhardtii mutant: an accumulation of pyropheophorbide a by anaerobiosis

Chlorophyll degradation was investigated in cells of a chlorophyll b-less mutant of Chlamydomonas reinhardtii under aerobic and anaerobic conditions. During degradation of chlorophyll under anaerobic conditions, chlorophyll catabolite P535, an open-tetrapyrrole, was not excreted, but pyropheophorbide a was accumulated as the end product with a transient accumulation of chlorophyllide a and pheophorbide a in cells, in contrast to the breakdown under aerobic conditions. It is likely that in the absence of oxygen, degradation of chlorophyll a proceeds to pyropheophorbide a by three consecutive reactions, dephytylation, metal-releasing and demethoxycarbonylation, and then stops due to a limitation of the oxygen that the monooxygenase reaction requires for bilin formation. A novel enzyme catalyzing demethoxycarbonylation of pheophorbide a was partially purified. The enzyme activity increased dependent on the age of cells, and its increase was completely suppressed by cycloheximide. Production of P535 was also dependent on cytoplasmic protein synthesis.     

Chlorophyll synthetase in chlorophyll-free chromoplasts

A considerable incorporation of [1-¹⁴C]isopentenyl diphosphate into chlorophyll in chromoplast preparations from daffodil flowers (Narcissus pseudonarcissus L.) was observed when exogenous chlorophyllide a was added. The enzyme chlorophyll synthetase showed properties of a peripheral membrane protein.Abbreviations IPP isopentenyl diphosphate - GGPP geranylgeranyl diphosphate - Chl_GG chlorophyll a esterified with geranylgeraniol - Chl_p chlorophyll a esterified with phytol - Chlide chlorophyllide a - HPLC high pressure liquid chromatography