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PubMed2002

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.

Avena↗
PubMed2002

Characterization of two phases of chlorophyll formation during greening of etiolated barley leaves.

The esterification kinetics of chlorophyllide, obtained by a single flash of light, were investigated in etiolated barley ( Hordeum vulgare L.) and oat ( Avena sativa L.) leaves. A rapid phase, leading to esterification of 15% of total chlorophyllide within 15-30 s, was followed by a lag-phase of nearly 2 min and a subsequent main phase, leading to esterification of 85% of total chlorophyllide within 30-60 min. The presence of additional protochlorophyllide, produced in the leaves by incubation with 5-aminolevulinate, did not change the esterification kinetics. The rapid phase was identical after partial (11-15%) and full (>80%) photoconversion of protochlorophyllide; the ability for a second rapid esterification phase was restored in a dark period of at least 10 min. Cooling the leaves to 0 degrees C abolished the esterification of the main phase while the rapid phase remained unchanged. The prolamellar bodies were already in part transformed into prothylakoid-like structures within 2-5 min after a full flash but not after a weak flash (11% photoconversion); in the latter case, the corresponding transformation required a dark period of about 45 min. The existence of subcomplexes of prolamellar bodies containing NADPH:protochlorophyllide oxidoreductase and chlorophyll synthase in the ratio 7:1 is discussed.

Aminolevulinic Acid↗
PubMed1985

Inactivation of chlorophyllase by negatively charged plant membrane lipids.

Chlorophyllide combines spontaneously not only with phosphatidylcholine (PC) liposomes but also with various other (plant) lipids dispersed in an aqueous medium. The lipid-associated chlorophyllide is highly fluorescent and the fluorescence yield is virtually independent of the nature of the lipid. Chlorophyllase (chlorophyll chlorophyllidohydrolase, EC 3.1.1.14) activity assays that are based on the determination of this chlorophyllide fluorescence show that phosphatidylglycerol (PG), and also sulphoquinovosyldiacylglycerol (SQDG), associate with isolated chlorophyllase, thereby inactivating the enzyme in a co-operative way. The extent of this inactivation depends on the pH and ionic strength of the reaction medium and can be completely reversed by divalent cations (Mg2+). The inhibition of chlorophyllase effected by free PG liposomes can be counteracted by electrically neutral lipids at relatively high concentration (PC and also chloroplast lipids). Digalactosyldiacylglycerol (DGDG) is not effective in this respect. When PG has been incorporated in PC or DGDG liposomes, its ability to inhibit chlorophyllase activity is reduced. Whereas the remaining chlorophyllase-inactivating effect of PG, incorporated in PC, can still be reversed by Mg2+, this is not found when enzyme inactivation is caused by PG incorporated in DGDG. The results reported here are consistent with those obtained earlier concerning the stabilization of chlorophyllase by PG and PG/galactolipid mixtures (Lambers, J.W.J., Verkleij, A.J. and Terpstra, W. (1984) Biochim. Biophys. Acta 786, 1-8). They are discussed in terms of the regulation of chlorophyllase activity by lipids surrounding the enzyme and by divalent cations.

Carboxylic Ester Hydrolases↗
PubMed2002

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.

Binding Sites↗
PubMed2001

On the nature of the two pathways in chlorophyll formation from protochlorophyllide.

The kinetics of formation of esterified chlorophyll in etiolated barley (Hordeum vulgare L.) leaves after illumination with a single flash was studied. It was found that after partial (14-24%) and after full photoreduction of protochlorophyllide, the same quantity of esterified products appear during the first 5 s after the flash. The rest of formed chlorophyllide was esterified in a slow process during at least 30 min at 15 degrees C. The product of fast esterification can be correlated with 'short-wavelength' chlorophyll, characterized by a fluorescence emission peak at 673-675 nm. This is the only chlorophyll form detectable within 20 s after partial (14%) photoconversion, and it appears at the same time as the shoulder of the chlorophyll(ide) fluorescence after full photoconversion. The main product after full photoconversion shows a fluorescence at 689 nm shifting in darkness within 15 s to 693 nm and then within 30 min to 682 nm (Shibata shift). The slow esterification proceeds with similar kinetics as the Shibata shift. We propose that the fast esterification of only part of total chlorophyllide after full photoconversion of protochlorophyllide in etiolated leaves reflects the restricted capacity of the esterifying system. The slow esterification of the residual chlorophyllide may be time-limited by its release from protochlorophyllide oxidoreductase, by disaggregation of prolamellar bodies and by diffusion of tetraprenyl diphosphates towards chlorophyll synthase.

Journal Article↗
PubMed2003

Phytol as one of the determinants of chlorophyll interactions in solution.

Optical absorption and fluorescence parameters of chlorophyll a and the phytol-free chlorophyllide a, as well as of their Mg-depleted derivatives, were compared in a series of organic solvents. In contrast to prevailing opinion, the spectral properties of chlorophyll are not indifferent to the removal of phytol. The electronic absorption spectra of chlorophyll a and chlorophyllide a differ and display a different dependence on the nature of the solvent, which cannot be explained solely by the location of a charged carboxylic group in the proximity of the pi- electron system. In fact, measurements in media of varying basicity show that deprotonation of the free carboxylic group in chlorophyllide, i.e., the presence of a negative point charge near the macrocycle, has no effect on pigment absorption spectra. Analysis of the solvent effect on the Q(Y) energies in terms of solvent polarity reveals that the phytyl moiety perturbs the spectral features of chlorophyll, mainly due to its interactions with the pigment solvation shell. The phytyl residue might also be thus partly involved in controlling the central metal ligation in chlorophylls. This influence of phytol on the spectral features of chlorophyll should be taken into account when comparing the spectra in solution with various spectral forms of chlorophyll in vivo.

Journal Article↗
PubMed1998

Chlorophyll synthesis in dark-grown pine primary needles

The pigment content of dark-grown primary needles of Pinus jeffreyi L. and Pinus sylvestris L. was determined by high-performance liquid chromatography. The state of protochlorophyllide a and of chlorophylls during dark growth were analyzed by in situ 77 K fluorescence spectroscopy. Both measurements unambiguously demonstrated that pine primary needles are able to synthesize chlorophyll in the dark. Norflurazon strongly inhibited both carotenoid and chlorophyll synthesis. Needles of plants treated with this inhibitor had low chlorophyll content, contained only traces of xanthophylls, and accumulated carotenoid precursors. The first form of chlorophyll detected in young pine needles grown in darkness had an emission maximum at 678 nm. Chlorophyll-protein complexes with in situ spectroscopic properties similar to those of fully green needles (685, 695, and 735 nm) later accumulated in untreated plants, whereas in norflurazon-treated plants the photosystem I emission at 735 nm was completely lacking. To better characterize the light-dependent chlorophyll biosynthetic pathway in pine needles, the 77 K fluorescence properties of in situ protochlorophyllide a spectral forms were studied. Photoactive and nonphotoactive protochlorophyllide a forms with emission properties similar to those reported for dark-grown angiosperms were found, but excitation spectra were substantially red shifted. Because of their lower chlorophyll content, norflurazon-treated plants were used to study the protochlorophyllide a photoreduction process triggered by one light flash. The first stable chlorophyllide photoproduct was a chlorophyllide a form emitting at 688 nm as in angiosperms. Further chlorophyllide a shifts usually observed in angiosperms were not detected. The rapid regeneration of photoactive protochlorophyllide a from nonphotoactive protochlorophyllide after one flash was demonstrated.

Journal Article↗
PubMed1980

Influence of Anaerobiosis on Chlorophyll Biosynthesis in Greening Oat Seedlings (Avena sativa L.).

The influence of anaerobiosis for 0.5 to 15 hours on the last steps of chlorophyll biosynthesis of etiolated oat seedlings was investigated. Phototransformation of protochlorophyllide to chlorophyllide is only slightly reduced and esterification of chlorophyllide is slightly increased by pretreatment under anaerobic conditions. Pretreated plants accumulate the geranylgeraniol ester of chlorophyllide rather than the phytol ester. Enzymic hydrogenation of the esterifying alcohol geranylgeraniol to phytol is presumably inhibited by anaerobiosis.

Journal Article↗
PubMed1992

The Shibata Shift and the Transformation of Etioplasts to Chloroplasts in Wheat with Clomazone (FMC 57020) and Amiprophos-Methyl (Tokunol M).

The Shibata shift is a change in the absorption maximum of chlorophyllide from 684 to 672 nanometers that occurs within approximately 0.5 hour of phototransformation of protochlorophyllide to chlorophyllide. Two compounds, clomazone and amiprophos-methyl, which previously have been shown to inhibit the Shibata shift in vivo, were used to look for correlations between the Shibata shift and other processes that occur during etioplast to chloroplast transformation. Leaf sections from 6-day-old etiolated wheat seedlings (Triticum aestivum L. cv Walde) were treated with 0.5 millimolar clomazone or 0.1 millimolar amiprophos-methyl in darkness. In addition to the Shibata shift, the esterification of chlorophyllide to chlorophyll and the relocation of protochlorophyllide reductase from the prolamellar bodies to the developing thylakoids were inhibited by these treatments. Prolamellar body transformation did not appear to be affected by amiprophos-methyl and was only slightly affected by clomazone. The results indicate that: (a) there is a strong correlation between the occurrence of the Shibata shift and esterification activity; (b) transformation of the prolamellar bodies does not depend on the Shibata shift; and (c) the occurrence of the Shibata shift may be a prerequisite to the relocation of protochlorophyllide reductase from prolamellar bodies to thylakoids.

Journal Article↗
PubMed1990

In vitro synthesis of chlorophyll a in the dark triggers accumulation of chlorophyll a apoproteins in barley etioplasts.

An in vitro translation system using lysed etioplasts was developed to test if the accumulation of plastid-encoded chlorophyll a apoproteins is dependent on the de novo synthesis of chlorophyll a. The P700 apoproteins, CP47 and CP43, were not radiolabeled in pulsechase translation assays employing lysed etioplasts in the absence of added chlorophyll precursors. When chlorophyllide a plus phytylpyrophosphate were added to lysed etioplast translation assays in the dark, chlorophyll a was synthesized and radiolabeled P700 apoproteins, CP47 and CP43, and a protein which comigrates with D1 accumulated. Chlorophyllide a or phytylpyrophosphate added separately to the translation assay in darkness did not induce chlorophyll a formation or chlorophyll a apoprotein accumulation. Chlorophyll a formation and chlorophyll a apoprotein accumulation were also induced in the lysed etioplast translation system by the photoreduction of protochlorophyllide to chlorophyllide a in the presence of exogenous phytylpyrophosphate. Accumulation of radiolabeled CP47 was detectable when very low levels of chlorophyll a were synthesized de novo (less than 0.01 nmol/10(7) plastids), and radiolabel increased linearly with increasing de novo chlorophyll a formation. Higher levels of de novo synthesized chlorophyll a were required prior to detection of radiolabel incorporation into the P700 apoproteins and CP43 (greater than 0.01 nmol/10(7) plastids). Radiolabel incorporation into the P700 apoproteins, CP47 and CP43, saturated at a chlorophyll a concentration which corresponds to 50% of the etioplast protochlorophyllide content (0.06 nmol of chlorophyll a/10(7) plastids).

Apoproteins↗
PubMed1995

Altered monovinyl and divinyl protochlorophyllide pools in bchJ mutants of Rhodobacter capsulatus. Possible monovinyl substrate discrimination of light-independent protochlorophyllide reductase.

In land plants in particular, it has been well established that chlorophyll intermediates, Mg-protoporphyrin, Mg-protoporphyrin monomethylester, protochlorophyllide, and chlorophyllide occur as monovinyl and divinyl forms. The pool of monovinyl and divinyl intermediates differ according to species, age of tissue, and light regime. In this study, we investigated the monovinyl and divinyl characteristics of protochlorophyllide and chlorophyllide in the purple non-sulfur photosynthetic eubacterium Rhodobacter capsulatus. Our results indicate that mutations in genes known to completely block the reduction of protochlorophyllide to chlorophyllide (such as bchN, bchB, and bchL mutants), accumulate a pool of monovinyl and divinyl forms of protochlorophyllide just as observed in plants. However, we also observed that directed insertion and deletion mutations in bchJ, a gene located in the photosynthesis gene cluster, affected the ratio of monovinyl and divinyl protochlorophyllide. Specifically, bchJ-disrupted strains accumulate reduced levels of bacteriochlorophyll concomitant with the accumulation of divinyl protochlorophyllide. Mutants of bchJ in combination with a second mutation in bchL still produce a mixed pool of monovinyl and divinyl protochlorophyllide; however, the ratio of monovinyl to divinyl protochlorophyllide is skewed in favor of divinyl protochlorophyllide. These results thus identify bchJ as the first sequenced gene that affects the divinyl to monovinyl ratio of photopigment intermediates in any photosynthetic organism. In addition, the results of our study also suggest that light-independent protochlorophyllide reductase is discriminatory for a monovinyl substrate.

Base Sequence↗
PubMed1995

Separation of mono- and divinyl chlorophyll species by high-performance liquid chromatography using an octadecyl polyvinyl alcohol polymer column.

Most of the chlorophylls and their related compounds from nonesterified chlorophyllide to esterified pheophytin were separated by high- performance liquid chromatography using a wide pore, C(18) polyvinyl alcohol polymer column with an elution using a binary gradient starting from a buffered mobile phase. The high selectivity of this system enabled not only the separation of common chlorophyll species but also the resolution of structurally similar compounds such as mono- and divinyl chlorophyllide and pheophorbide species which usually coelute on monomeric bonded phases. The method is successfully applied to analyses of the pigments extracted from green and etiolated tissues of higher plants and photosynthetic bacterial cells.

Chlorophyll↗
PubMed2003

Activation parameters of the blue shift (Shibata shift) subsequent to protochlorophyllide phototransformation.

The Shibata shift was analyzed in flash irradiated wheat (Triticum aestivum, L., cult. MV17) leaf homogenates in the pressure range of 0.1 to 500 MPa, at temperatures of 20, 30 and 40 degrees C. The kinetics of the blue shift (called Shibata shift in case of intact leaves) was followed by repeated recording of fluorescence emission spectra after phototransformation. At 20 degrees C, above 100 MPa, the blue shift slowed down remarkably. Two components of the blue shift could be distinguished, one was pressure-dependent and the other was almost pressure-independent. The pressure-independent component can be associated with minor conformational changes of the NADPH:protochlorophyllide oxidoreductase (POR) enzyme, followed by molecular movements of the newly formed chlorophyllide molecules. The calculated activation volume of the pressure-dependent component was 43+/-11 cm(3) mol(-1) at 20 degrees C. This value reflects major molecular reorganizations in the lipid system of the membrane and in the chlorophyllide-protein complexes, and corresponds to changes of the tertiary structure of proteins which can proceed directly or indirectly via structural changes of the membrane lipids. The process was inhibited by 300 and 400 MPa at 30 and 40 degrees C, respectively. The activation volume reduced to 35+/-1.5 cm(3) mol(-1) at 40 degrees C. The decrease of the activation volume with increasing temperature indicates that the blue shift requires loosened lipid structures. The activation energy of the blue shift (measured between 10 and 40 degrees C at atmospheric pressure) was 100+/-20 kJ/mol, indicating that the structural change involves rearrangement of strong molecular interactions.

Chlorophyllides↗
PubMed2007

Chloroplast biogenesis: the use of mutants to study the etioplast-chloroplast transition.

In angiosperm plants, the etioplast-chloroplast transition is light-dependent. A key factor in this process is the protochlorophyllide oxidoreductase A (PORA), which catalyzes the light-induced reduction of protochlorophyllide to chlorophyllide. The import pathway of the precursor protein prePORA into chloroplasts was analyzed in vivo and in vitro by using homozygous loss-of-function mutants in genes coding for chlorophyllide a oxygenase (CAO) or for members of the outer-envelope solute-channel protein family of 16 kDa (OEP16), both of which have been implied to be key factors for the import of prePORA. Our in vivo analyses show that cao or oep16 mutants contain a normally structured prolamellar body that contains the protochlorophyllide holochrome. Furthermore, etioplasts from cao and oep16 mutants contain PORA protein as found by mass spectrometry. Our data demonstrate that both CAO and OEP16 are dispensable for chloroplast biogenesis and play no central role in the import of prePORA in vivo and in vitro as further indicated by protein import studies.

Arabidopsis↗
PubMed1995

Enzymatic product formation impairs both the chloroplast receptor-binding function as well as translocation competence of the NADPH: protochlorophyllide oxidoreductase, a nuclear-encoded plastid precursor protein.

The key enzyme of chlorophyll biosynthesis in higher plants, the light-dependent NADPH:protochlorophyllide oxidoreductase (POR, EC 1.6.99.1), is a nuclear-encoded plastid protein. Its posttranslational transport into plastids of barley depends on the intraplastidic availability of one of its substrates, protochlorophyllide (PChlide). The precursor of POR (pPOR), synthesized from a corresponding full-length barley cDNA clone by coupling in vitro transcription and translation, is enzymatically active and converts PChlide to chlorophyllide (Chlide) in a light- and NADPH-dependent manner. Chlorophyllide formed catalytically remains tightly but noncovalently bound to the precursor protein and stabilizes a transport-incompetent conformation of pPOR. As shown by in vitro processing experiments, the chloroplast transit peptide in the Chlide-pPOR complex appears to be masked and thus is unable to physically interact with the outer plastid envelope membrane. In contrast, the chloroplast transit peptide in the naked pPOR (without its substrates and its product attached to it) and in the pPOR-substrate complexes, such as pPOR-PChlide or pPOR-PChlide-NADPH, seems to react independently of the mature region of the polypeptide, and thus is able to bind to the plastid envelope. When envelope-bound pPOR-PChlide-NADPH complexes were exposed to light during a short preincubation, the enzymatically produced Chlide slowed down the actual translocation step, giving rise to the sequential appearance of two partially processed translocation intermediates. However, ongoing translocation induced by feeding the chloroplasts delta-aminolevulinic acid, a precursor of PChlide, was able to override these two early blocks in translocation, suggesting that the plastid import machinery has a substantial capacity to denature a tightly folded, envelope-bound precursor protein. Together, our results show that pPOR with Chlide attached to it is impaired both in the ATP-dependent step of binding to a receptor protein component of the outer chloroplast envelope membrane, as well as in the PChlide-dependent step of precursor translocation.

Biological Transport, Active↗
PubMed1980

Detection and partial characterization of activity of chlorophyll synthetase in etioplast membranes.

The esterification of chlorophyllide a was investigated an irradiated etioplast-membrane fractions ('broken etioplasts') from oat seedlings (Avena sativa L.). As a substrate, [1(-3)H]geranylgeraniol and its monophosphate and diphosphate derivatives were prepared by chemical synthesis. Geranylgeraniol and its monphosphate derivative are incorporated into chlorophyll only in the presence of ATP whereas the diphosphate derivative is incorporated also without ATP. The yield of esterified chlorophyll is 80-90% of chlorophyllide with saturating substrate concentrations. The term 'chlorophyll synthetase' is used to describe the enzyme activity which is different from chlorophyllase. Other substrates are phytol and farnesol either with ATP or as the diphosphate derivatives. The relative specificity of 'chlorophyll synthetase' for thse substrates is geranylgeraniol:phytol:farnesol = 6:3:1. In these experiments in vitro, a new chlorphyll esterified with farnesol was detected which does not occur in intact plants. Geraniol and n-pentadecanol are no substrates for the enzyme. Protochlorphyllide which is present in non-irradiated etioplast membrane fractions is not esterified under the same conditions.

Carbon-Oxygen Ligases↗
PubMed2002

Catalytic function of a novel protein protochlorophyllide oxidoreductase C of Arabidopsis thaliana.

In Arabidopsis thaliana Por C has been identified only on sequence homology to that of por A and por B. To demonstrate its catalytic function Arabidopsis thaliana protochlorophyllide oxidoreductase C gene (por c) that codes for the mature part of POR C protein having 335 amino acids was expressed in Escherchia coli cells. The POR C enzyme in the presence of NADPH and protochlorophyllide when incubated in dark formed a ternary complex. When it was excited at 433 nm, it had a fluorescence emission peak at 636 nm. After illumination with actinic cool white fluorescent light, a peak at 673 nm due to chlorophyllide gradually increased with concomitant decrease of 636 nm emission, demonstrating the gradual phototransformation of protochlorophyllide to chlorophyllide. The significance of differential por gene expression in light and dark among different species is discussed.

Arabidopsis↗
PubMed1993

Genetic analysis of the bchC and bchA genes of Rhodobacter sphaeroides.

This study has identified by sequence analysis a single gene in the bchC locus of Rhodobacter sphaeroides and three genes, designated bchX, Y and Z, in the bchA locus, which was previously thought to contain only a single gene. All four genes may reside within the same operon and are transcribed in the order bchC-X-Y-Z. Complementation analysis of eight transposon insertion mutants within these genes suggests that bchX, Y and Z are essential for the reduction of 2-devinyl-2-hydroxyethyl chlorophyllide a and that bchC encodes the 2-desacetyl-2-hydroxyethyl bacteriochlorophyllide a dehydrogenase. Similarity between the putative BchX protein and dinitrogenase reductase proteins suggests that BchX may also be a reductase, supplying electrons for reduction of 2-devinyl-2-hydroxyethyl chlorophyllide a.

Amino Acid Sequence↗