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At least 19 recordsLinked to original sources
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↗
PubMed1973

The Conversion of Photoinactive Protochlorophyllide(633) to Phototransformable Protochlorophyllide(650) in Etiolated Bean Leaves Treated with delta-Aminolevulinic Acid.

The relationship of phototransformable protochlorophyllide to photoinactive protochlorophyllide has been studied in primary leaves of 7- to 9-day-old dark-grown bean (Phaseolus vulgaris L. var. Red Kidney) seedlings. Various levels of photoinactive protochlorophyllide, absorbing at 633 nm in vivo, were induced by administering delta-aminolevulinic acid to the leaves in darkness. Phototransformable protochlorophyllide, absorbing at 650 nm in vivo, was subsequently transformed to chlorophyllide by a light flash, and the regeneration of the photoactive pigment was followed by monitoring the absorbance increase at 650 nm in vivo. A small increase in the level of protochlorophyllide(633) causes a marked increase in the extent of regeneration of protochlorphyllide(650) following a flash. High levels of the inactive pigment species, however, retard the capacity to reform photoactive protochlorophyllide. A nonstoichiometric and kinetically complex decrease in absorbance at 633 nm in vivo accompanied the absorbance increase at 650 nm. The half-time for protochlorophyllide(650) regeneration in control leaves was found to be three times longer than the half-time for conversion of chlorophyllide(678) to chlorophyllide(683) at 22 C. The results are consistent with the hypothesis that protochlorophyllide(633) is a direct precursor of protochlorophyllide(650) and that the protein moiety of the protochlorophyllide holochrome acts as a "photoenzyme" in the conversion of protochlorophylide to chlorophyllide.

Journal Article↗
PubMed1993

Phototransformation of monovinyl and divinyl protochlorophyllide by NADPH:protochlorophyllide oxidoreductase of barley expressed in Escherichia coli.

The chlorophyll precursors monovinyl protochlorophyllide (MV-PChlide) and divinyl protochlorophyllide (DV-PChlide) were extracted from mutant C-2A' of the unicellular green alga Scenedesmus obliquus which accumulates both protochlorophyllide derivatives in the dark. The two pigments were characterized by absorption and fluorescence spectroscopy and by plasma desorption mass spectrometry. The molecular masses of MV-PChlide and DV-PChlide were determined as 612 and 610 atomic mass units (amu) respectively. Both MV-PChlide and DV-PChlide were accepted as substrates and photoconverted to chlorophyllides in vitro by NADPH:protochlorophyllide oxidoreductase of barley expressed in Escherichia coli.

Chlorophyta↗
PubMed1988

Chloroplast Biogenesis 60 : Conversion of Divinyl Protochlorophyllide to Monovinyl Protochlorophyllide in Green(ing) Barley, a Dark Monovinyl/Light Divinyl Plant Species.

In higher plants, most of the chlorophyll a is formed via the divinyl and monovinyl chlorophyll monocarboxylic biosynthetic routes. These two routes are strongly interconnected prior to protochlorophyllide formation in barley (Hordeum vulgare L. cv Morex), a dark monovinyl-light divinyl plant species, but not in cucumber (Cucumis sativus L. cv Beit Alpha MR), a dark divinyl-light divinyl plant species (BC Tripathy, CA Rebeiz, 1986 J Biol Chem 261: 13556-13564). It is shown that in dark monovinyl-light divinyl plant species such as barley, the divinyl and monovinyl monocarboxylic routes become interconnected at the level of protochlorophyllide during transition from the divinyl to the monovinyl protochlorophyllide biosynthetic mode. In cucumber, a dark divinyl-light divinyl plant species, in which the monovinyl monocarboxylic biosynthetic route becomes preponderant only after an abnormally long sojourn in darkness, the conversion of divinyl to monovinyl protochlorophyllide does not take place on the barley time-scale of incubation.

Journal Article↗
PubMed1996

Protochlorophyllide reductase. III: Synthesis of a protochlorophyllide-dihydroflavin complex.

A mild and efficient method of linking a dihydroflavin to the C-17 carboyxlic acid side chain of protochlorophyllide, without degradation of the sensitive E ring or loss of magnesium, is described. The appended dihydroflavin was shown to quench the fluorescence of protochlorophyllide. In contrast, a dihydronicotinamide moiety was unable to effect fluorescence quenching. the relevance of these findings to a possible mechanism of action of the enzyme protochlorophyllide reductase is discussed.

Flavins↗
PubMed1996

The light intensity dependence of protochlorophyllide photoconversion and its significance to the catalytic mechanism of protochlorophyllide reductase.

The light-dependent step in chlorophyll synthesis by higher plants involves hydrogen transfer from NADPH+ to the porphyrin protochlorophyllide catalysed by the photoenzyme protochlorophyllide reductase. The light intensity dependence of the process has been studied in vitro using wheat etioplast membranes. The results suggest that a single photochemical event is involved in the photoconversion. In support of this conclusion we also demonstrate that illumination of these membranes with a train of ultrashort (150 fs) flashes resulted in chlorophyll accumulation. The significance of the findings in terms of possible mechanisms for the reductase are discussed.

Catalysis↗
PubMed1999

Protochlorophyllide oxidoreductase B-catalyzed protochlorophyllide photoreduction in vitro: insight into the mechanism of chlorophyll formation in light-adapted plants.

The mechanism of the protochlorophyllide (PChlide) photoreduction reaction operating in light-adapted plants and catalyzed by NADPH:protochlorophyllide oxidoreductase B (PORb) has been analyzed by low-temperature fluorescence spectroscopy by using purified barley PORb overexpressed heterologously in Escherichia coli as a fusion protein with the maltose-binding protein. We show that the PORb-catalyzed PChlide reduction reaction consists of two steps, one photochemical and the other nonphotochemical. The initial photochemical reaction follows a single quantum mechanism and leads to the formation of an unstable intermediate with mixed pigment electronic structure and an EPR spectrum that suggests the presence of a free electron. The second step involves the spontaneous conversion of the unstable intermediate into chlorophyllide as defined by its spectroscopic characteristics and migration on an HPLC column. Both steps of the reaction can be performed at subzero temperatures in frozen samples, suggesting that they do not include major changes in enzyme conformation or pigment rearrangement within the active site. The rate of the reaction at room temperature depends linearly on enzyme and substrate (PChlide) concentration, and the kinetic parameters are consistent with one molecule of substrate bound per active monomer in solution. The PORb-catalyzed reaction in vitro is spectroscopically similar to that identified in leaves of light-adapted plants, suggesting that the same reaction sequence observed operates in planta.

ATP-Binding Cassette Transporters↗
PubMed2002

In situ conversion of protochlorophyllide b to protochlorophyllide a in barley. Evidence for a novel role of 7-formyl reductase in the prolamellar body of etioplasts.

We recently put forth a model of a protochlorophyllide (Pchlide) light-harvesting complex operative during angiosperm seedling de-etiolation (Reinbothe, C., Lebedev, N., and Reinbothe, S. (1999) Nature 397, 80-84). This model, which was based on in vitro reconstitution experiments with zinc analogs of Pchlide a and Pchlide b and the two NADPH:protochlorophyllide oxidoreductases (PORs), PORA and PORB, of barley, predicted a 5-fold excess of Pchlide b, relative to Pchlide a, in the prolamellar body of etioplasts. Recent work (Scheumann, V., Klement, H., Helfrich, M., Oster, U., Schoch, S., and Rüdiger, W. (1999) FEBS Lett. 445, 445-448), however, contradicted this model and reported that Pchlide b would not be present in etiolated plants. Here we demonstrate that Pchlide b is an abundant pigment in barley etioplasts but is rather metabolically unstable. It is rapidly converted to Pchlide a by virtue of 7-formyl reductase activity, an enzyme that had previously been implicated in the chlorophyll (Chl) b to Chl a reaction cycle. Our findings suggest that etiolated plants make use of 7-formyl reductase to fine tune the levels of Pchlide b and Pchlide a and thereby may regulate the steady-state level of light-harvesting POR-Pchlide complex.

Chlorophyll↗
PubMed2002

In vitro reconstitution of light-harvesting POR-protochlorophyllide complex with protochlorophyllides a and b.

NADPH:protochlorophyllide oxidoreductase (POR; EC ) is a key enzyme for the light-induced greening of angiosperms. In barley, two POR proteins exist, termed PORA and PORB. These have previously been proposed to form higher molecular weight light-harvesting complexes in the prolamellar body of etioplasts (Reinbothe, C., Lebedev, N., and Reinbothe, S. (1999) Nature 397, 80-84). Here we report the in vitro reconstitution of such complexes from chemically synthesized protochlorophyllides (Pchlides) a and b and galacto- and sulfolipids. Low temperature (77 K) fluorescence measurements revealed that the reconstituted, lipid-containing complex displayed the same characteristics of photoactive Pchlide 650/657 as the presumed native complex in the prolamellar body. Moreover, Pchlide F650/657 was converted to chlorophyllide (Chlide) 684/690 upon illumination of the reconstituted complex with a 1-ms flash of white light. Identification and quantification of acetone-extractable pigments revealed that only the PORB-bound Pchlide a had been photoactive and was converted to Chlide a, whereas Pchlide b bound to the PORA remained photoinactive. Nondenaturing PAGE of the reconstituted Pchlide a/b-containing complex further demonstrated a size similar to that of the presumed native complex in vivo, suggesting that both complexes may be identical.

Blotting, Western↗
PubMed1980

The protochlorophyllide holochrome of barley (Hordeum vulgare L.). Isolation and characterization of the NADPH:protochlorophyllide oxidoreductase.

The NADPH:protochlorophyllide oxidoreductase of barley has been solubilized from etioplast membranes and purified to apparent homogeneity. The highest specific activity measured for the purified enzyme was 1.6 nmol chlorophyllide formed (mg protein-1) per flash. Electrophoretic analysis of the purified enzyme on sodium dodecylsulfate/polyacrylamide gels revealed only one polypeptide of Mr 36000. Durig glycerol gradient centrifugation the enzyme migrates as a low-molecular-weight component. It is proposed that each enzyme molecule contains only one polypeptide chain. Assuming a molecular weight of 36000 for the enzyme, it was calculated that two or three protochlorophyllide molecules are bound to each enzyme molecule.

Chlorophyll↗
PubMed1993

Light-independent and light-dependent protochlorophyllide-reducing activities and two distinct NADPH-protochlorophyllide oxidoreductase polypeptides in mountain pine (Pinus mugo).

Lower plants and gymnosperms synthesize chlorophyll and develop photosynthetically competent chloroplasts even when grown in the dark. In cell-free extracts of pine (Pinus mugo, Turra, ssp. mugo) seedlings, light-independent and light-dependent protochlorophyllide-reducing activities are present. Two distinct NADPH-protochlorophyllide-oxidoreductase (POR) polypeptides can be detected immunologically with an antiserum raised against the POR of barley. The subcellular localization and amounts of the two POR polypeptides are differentially affected by light: one of them is predominantly present in prolamellar bodies of etiochloroplasts and its abundance rapidly declines once the pine seedlings are exposed to light; the other is found in thylakoid membranes and its amount does not change during illumination of dark-grown seedlings. Two types of cDNA sequences are identified that encode two distinct POR polypeptides in pine. The relevance of these POR polypeptides for the two chlorophyll biosynthetic pathways active in gymnosperms is discussed.

Amino Acid Sequence↗
PubMed2006

In vitro-mutagenesis of NADPH:protochlorophyllide oxidoreductase B: two distinctive protochlorophyllide binding sites participate in enzyme catalysis and assembly.

NADPH:protochlorophyllide oxidoreductase (POR) B is a key enzyme for the light-induced greening of etiolated angiosperm plants. It is nucleus-encoded, imported into the plastids posttranslationally, and assembled into larger light-harvesting POR:protochlorophyllide complexes termed LHPP (Reinbothe et al., Nature 397:80-84, 1999). An in vitro-mutagenesis approach was taken to study the role of the evolutionarily conserved Cys residues in pigment binding. Four Cys residues are present in the PORB of which two, Cys276 and Cys303, established distinct pigment binding sites, as shown by biochemical tests, protein import studies, and in vitro-reconstitution experiments. While Cys276 constituted the Pchlide binding site in the active site of the enzyme, Cys303 established a second, low affinity pigment binding site that was involved in the assembly and stabilization of imported PORB enzyme inside etioplasts.

Amino Acid Sequence↗
PubMed2001

Both light-dependent protochlorophyllide oxidoreductase A and protochlorophyllide oxidoreductase B are down-regulated in the slender mutant of barley.

The gibberellin-insensitive overgrowth mutant of barley, slender, exhibits altered expression of a number of nuclear genes in comparison with the wild type. There is a particularly marked reduction in slender seedlings of transcript encoding protochlorophyllide oxidoreductase (POR), the enzyme which catalyses the penultimate and only light-requiring step in chlorophyll biosynthesis. The expression of the two barley genes encoding light-dependent POR, PORA and PORB was investigated. Expression of both genes was found to be reduced in slender seedlings relative to the wild type, in both etiolated and light-grown leaf tissue; this was most marked in the zone of rapid cell extension. Western blot analysis showed that POR protein was also less abundant in etiolated and in light-grown slender than in the equivalent wild-type leaf tissue, although the effect was less pronounced than at the transcript level. Protochlorophyllide content in etiolated slender seedlings was reduced in comparison with wild-type seedlings, though chlorophyll content in light-grown leaf blades was unaffected. The reduction in POR expression in slender barley may reflect a novel response to the constitutive activation of gibberellin signalling in this mutant. Despite the consequences of the mutation for POR gene expression, slender seedlings develop apparently normal chloroplasts in the light, and etioplasts with well-defined prolamellar bodies when grown in continuous darkness. This suggests that the POR content of wild-type barley seedlings is well in excess of the minimum required for normal plastid development.

Blotting, Northern↗
PubMed2005

A role of Toc33 in the protochlorophyllide-dependent plastid import pathway of NADPH:protochlorophyllide oxidoreductase (POR) A.

NADPH:protochlorophyllide oxidoreductase (POR) A is a key enzyme of chlorophyll biosynthesis in angiosperms. It is nucleus-encoded, synthesized as a larger precursor in the cytosol and imported into the plastids in a substrate-dependent manner. Plastid envelope membrane proteins, called protochlorophyllide-dependent translocon proteins, Ptcs, have been identified that interact with pPORA during import. Among them are a 16-kDa ortholog of the previously characterized outer envelope protein Oep16 (named Ptc16) and a 33-kDa protein (Ptc33) related to the GTP-binding proteins Toc33 and Toc34 of Arabidopsis. In the present work, we studied the interactions and roles of Ptc16 and Ptc33 during pPORA import. Radiolabeled Ptc16/Oep16 was synthesized from a corresponding cDNA and imported into isolated Arabidopsis plastids. Crosslinking experiments revealed that import of 35S-Oep16/Ptc16 is stimulated by GTP. 35S-Oep16/Ptc16 forms larger complexes with Toc33 but not Toc34. Plastids of the ppi1 mutant of Arabidopsis lacking Toc33, were unable to import pPORA in darkness but imported the small subunit precursor of ribulose-1,5-bisphosphate carboxylase/oxygenase (pSSU), precursor ferredoxin (pFd) as well as pPORB which is a close relative of pPORA. In white light, partial suppressions of pSSU, pFd and pPORB import were observed. Our results unveil a hitherto unrecognized role of Toc33 in pPORA import and suggest photooxidative membrane damage, induced by excess Pchlide accumulating in ppi1 chloroplasts because of the lack of pPORA import, to be the cause of the general drop of protein import.

Amino Acid Sequence↗
PubMed1981

The protochlorophyllide holochrome of barley (Hordeum vulgare L.). Phytochrome-induced decrease of translatable mRNA coding for the NADPH: protochlorophyllide oxidoreductase.

During the illumination of dark-grown barley plants light induces a rapid decrease of a translatable mRNA which codes for a polypeptide of Mr 44000. This component was identified as a precursor of the NADPH:protochlorophyllide oxidoreductase. The precursor has an Mr larger than the authentic protein by approximately 8000. The light-induced change in the level of translatable mRNA can be induced by a 15-s red-light pulse followed by 5 h of darkness. The red-light effect is reversed by a subsequent far-red-light treatment. It is concluded that the light-induced decline of translatable mRNA for the NADPH:protochlorophyllide oxidoreductase is controlled by phytochrome. The significance of this finding for present concepts of light-dependent control of chloroplast development and chlorophyll synthesis is discussed.

Electrophoresis, Polyacrylamide Gel↗
PubMed1981

The protochlorophyllide holochrome of barley (Hordeum vulgare L.). The effect of light on the NADPH:protochlorophyllide oxidoreductase.

During the illumination of etiolated barley plants a rapid decline of the NADPH: protochlorophyllide oxidoreductase is observed. Within the first 5 min of continuous light approximately 90% of the enzyme activity present in dark-grown barley plants disappears and, at the same time, the amount of enzyme protein is diminished by more than 60%. No stable polypeptide fragments have been found which might be formed during the light-induced degradation of the enzyme protein. The rate of enzyme protein synthesis is not drastically affected at the beginning of the illumination period. During the subsequent light-dependent chloroplast development a phytochrome-induced decline in the rate of protein synthesis, concomittant with a continuous light-dependent degradation of the enzyme protein, leads to a progressive decrease of the concentration of the enzyme. After 6 h of continuous light, when the rate of chlorophyll accumulation is at its greatest, only traces of the enzyme protein are visible and the enzyme activity is no longer detectable within the plants. In contrast to previous concepts of chlorophyll biosynthesis in higher plants, our results present evidence that the NADPH: protochlorophyllide oxidoreductase functions only for a short time period after the onset of light.

Darkness↗
PubMed1998

Cloning of the gene encoding a protochlorophyllide reductase: the physiological significance of the co-existence of light-dependent and -independent protochlorophyllide reduction systems in the cyanobacterium Plectonema boryanum.

Cyanobacteria have two protochlorophyllide (Pchlide) reductases catalyzing the conversion of Pchlide to chlorophyllide, a key step in the biosynthetic pathway of chlorophylls (Chls); a light-dependent (LPOR) and a light-independent (DPOR) reductase. We found an open reading frame (ORF322) in a 2,131-bp EcoRI fragment from the genomic DNA of the cyanobacterium Plectonema boryanum. Because the deduced amino acid sequence showed a high similarity to those of various plant LPORs and the LPOR activity was detected in the soluble fraction of Escherichia coli cells over-expressing the ORF322 protein, ORF322 was defined as the por gene encoding LPOR in P. boryanum. A por-disrupted mutant, YFP12, was isolated by targeted mutagenesis to investigate the physiological importance of LPOR. YFP12 grew as well as wild type under low light conditions (10-25 muE m-2 s-1). However, its growth was significantly retarded as a result of a significant decrease in its Chl content under higher light conditions (85-130 muE m-2 s-1). Furthermore, YFP12 stopped growing and suffered from photobleaching under the highest light intensity (170 muE m-2 s-1). In contrast, a chlL-disrupted (DPOR-less) mutant YFC2 grew as well as wild type irrespective of light intensity. From these phenotypic characteristics, we concluded that, although both LPOR and DPOR contribute to Chl synthesis in the cells growing in the light, the extent of the contribution by LPOR increases with increasing light intensity; without it, the cells are unable to grow under light intensities of more than 130 muE m-2 s-1.

Amino Acid Sequence↗
PubMed2003

Fluorescence lifetimes of protochlorophyllide in plants with different proportions of short-wavelength and long-wavelength protochlorophyllide spectral forms.

Dark-grown leaves of maize (Zea mays), wheat (Triticum aestivum), wild-type pea (Pisum sativum) and its light-independent photomorphogenesis mutant (lip1) have different proportions of protochlorophyllide (Pchlide) forms as revealed by low-temperature fluorescence emission spectra. Four discrete spectral forms of Pchlide, with emission peaks around 633, 640, 656 and 670 nm, could be distinguished after Gaussian deconvolution. In maize and wheat the 656 nm component was the most prominent, whereas for wild-type pea and its lip1 mutant, the 633 and 640 nm components contributed mostly to the fluorescence emission spectra. For the fluorescence lifetimes measured at 77 K a double exponential model was the most adequate to describe the Pchlide fluorescence decay not only for the Pchlide(650-656) form but also for the short-wavelength Pchlide forms. A fast component in the range 0.3-0.8 ns and a slow component in the range 5.1-7.1 ns were present in all samples, but the values varied, depending on species. The long-wavelength Pchlide(650-656) form had a slow component with a lifetime between 5.1 and 6.7 ns, probably reflecting the fluorescence from aggregated Pchlide. The short-wavelength Pchlide(628-633) form had values of the slow component varying between 6.2 and 7.1 ns. This represents a monomeric but probably protein-bound Pchlide form because the free Pchlide in solution has a much longer lifetime around 10 ns at 77 K. The contribution of different Pchlide forms to the measured lifetime values is discussed.

Light↗