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PubMed2003

Functional analysis of isoforms of NADPH: protochlorophyllide oxidoreductase (POR), PORB and PORC, in Arabidopsis thaliana.

NADPH:protochlorophyllide oxidoreductase (POR) catalyzes the light-dependent reduction of protochlorophyllide. To elucidate the physiological function of three differentially regulated POR isoforms (PORA, PORB and PORC) in Arabidopsis thaliana, we isolated T-DNA tagged null mutants of porB and porC. The mature seedlings of the mutants had normal photosynthetic competencies, showing that PORB and PORC are interchangeable and functionally redundant in developed plants. In etiolated seedlings, only porB showed a reduction in the photoactive protochlorophyllide and the size of prolamellar bodies (PLBs), indicating that PORB, as well as PORA, functioned in PLB assembly and photoactive protochlorophyllide formation in etiolated seedlings. When illuminated, the etiolated porB seedling was able to green to a similar extent as the wild type, whereas the greening was significantly reduced under low light conditions. During greening, high light irradiation increased the level of PORC protein, and the greening of porC was repressed under high light conditions. The porB, but not porC, etiolated seedling was more sensitive to the far-red block of greening than the wild type, which is caused by depletion of endogenous POR proteins resulting in photo-oxidative damage. These results suggest that, at the onset of greening, PLBs are important for efficient capture of light energy for photoconversion under various light conditions, and PORC, which is induced by high light irradiation, contributes to photoprotection during greening of the etiolated seedlings.

Arabidopsis↗
PubMed1967

Studies on the regeneration of protochlorophyllide after brief illumination of etiolated bean leaves.

The effects of various inhibitors of nucleic acid and protein synthesis on protochlorophyllide synthesis in dark-grown Phaseolus vulgaris var. Red Kidney have been studied. Actinomycin D, chloramphenicol, and puromycin inhibit the regeneration of protochlorophyllide holochrome (detected as a 650 mmu absorption peak) in vivo in darkness after photoconversion of endogenous protochlorophyllide a to chlorophyllide a; this inhibition does not occur in similarly treated leaves supplied with delta-aminolevulinic acid.These data suggest that the regeneration of protochlorophyllide results from the synthesis of RNA and enzymes required for the production of delta-aminolevulinate.

Journal Article↗
PubMed1971

The sites of photoconversion of protochlorophyllide to chlorophyllide in barley seedlings.

The photoreduction of protochlorophyllide a to chlorophyllide a in intact 6-day-old seedlings of etiolated barley (Hordeum vulgare) exhibits a small initial phase, followed by an induction period of about 1 hour before a rapid phase of additional chlorophyll formation begins. Cycloheximide, an inhibitor of protein synthesis, has no effect on the initial phase of conversion of preformed protochlorophyllide, but it either abolishes or severely inhibits the subsequent phase of rapid chlorophyll synthesis within 45 minutes of its application to the seedlings. An analysis of the biphasic inhibition process suggests that the lifetime of the enzyme controlling protochlorophyllide synthesis (probably delta-amino-levulinic acid synthetase) is not longer than 10 minutes.The rapid phase of chlorophyll formation can be effected by a series of brief (15 second) pulses of light spaced at least 5 minutes apart. When longer dark intervals are used, no increase is observed in the yield of chlorophyll per pulse. We interpret the findings to indicate that the photoconversion takes place at distinct enzymatic sites whose concentration does not increase during a period of 4 hours following the initial illumination. The sites can be used repeatedly with a turnover time determined by the removal of the product chlorophyllide and the synthesis and placement of a new protochlorophyllide molecule.

Journal Article↗
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↗
PubMed1979

Kinetics of photoconversion of protochlorophyllide 649 to chlorophyllide 676 at low temperature in etiolated cotyledons of Pharbitis nil.

The kinetics of the photoconversion of protochlorophyllide 649 to chlorophyllide 676 were studied spectrophotometrically over the temperature range of -15 -- -80 degrees C under light-saturating conditions in etiolated cotyledons of Pharbitis nil. Photoconversion obeyed the sum of two first-order kinetics over this low temperature range. Activation energies obtained from the rate constants were about 5000 cal; this suggests that these two processes may be physical processes not chemical reactions. The results indicate that photoconversion involves two main steps. One is the step dependent on both light intensity and temperature that has been well studied. The other, which is concerned in this study, is the step dependent on temperature only, which may be the requisite for photoconversion. This latter step seems to be related to the binding mode of protochlorophyllide to a holochrome protein or to conformational changes in the protochlorophyllide-holochrome.

Chlorophyll↗
PubMed2002

Activation volumes of processes linked to the phototransformation of protochlorophyllide determined by fluorescence spectroscopy at high pressure.

The photochemical activity of NADPH:protochlorophyllide oxidoreductase (POR) was studied in etiolated wheat (Triticum aestivum, L., cult. MV17) leaf homogenates. The kinetics of the transformation of protochlorophyllide into chlorophyllide was detected by fluorescence intensity changes at 690 nm (formation of chlorophyllide) and 655 nm (decay of protochlorophyllide) at 20 degrees C, excited at 440 nm while the pressure was varied between 0.1 and 400 MPa. Both kinetics could be fitted by two exponentials and the reaction rates were pressure-dependent. A model was suggested based on the comparison of the two kinetics. Reaction rates of the processes occurring during the prototransformation were determined in function of pressure. The evaluation yielded the activation volume as 1.7 ml mol(-1), which corresponds with the formation of one H-bond/molecule.

Kinetics↗
PubMed2001

Tyr275 and Lys279 stabilize NADPH within the catalytic site of NADPH:protochlorophyllide oxidoreductase and are involved in the formation of the enzyme photoactive state.

Fluorescence spectroscopic and kinetic analysis of photochemical activity, cofactor and substrate binding, and enzyme denaturation studies were performed with highly purified, recombinant pea NADPH:protochlorophyllide oxidoreductase (POR) heterologously expressed in Escherichia coli. The results obtained with an individual stereoisomer of the substrate [C8-ethyl-C13(2)-(R)-protochlorophyllide] demonstrate that the enzyme photoactive state possesses a characteristic fluorescence maximum at 646 nm that is due to the presence of specific charged amino acids in the enzyme catalytic site. The photoactive state is converted directly into an intermediate having fluorescence at 685 nm in a reaction involving direct hydrogen transfer from the cofactor (NADPH). Site-directed mutagenesis of the highly conserved Tyr275 (Y275F) and Lys279 (K279I and K279R) residues in the enzyme catalytic pocket demonstrated that the presence of these two amino acids in the wild-type POR considerably increases the probability of photoactive state formation following cofactor and substrate binding by the enzyme. At the same time, the presence of these two amino acids destabilizes POR and increases the rate of enzyme denaturation. Neither Tyr275 nor Lys279 plays a crucial role in the binding of the substrate or cofactor by the enzyme. In addition, the presence of Tyr275 is absolutely necessary for the second step of the protochlorophyllide reduction reaction, "dark" conversion of the 685 nm fluorescence intermediate and the formation of the final product, chlorophyllide. We propose that Tyr275 and Lys279 participate in the proper coordination of NADPH and PChlide in the enzyme catalytic site and thereby control the efficiency of the formation of the POR photoactive state.

Amino Acid Substitution↗
PubMed1989

Nucleotide sequence of a cDNA coding for the NADPH-protochlorophyllide oxidoreductase (PCR) of barley (Hordeum vulgare L.) and its expression in Escherichia coli.

The primary structure of the NADPH-protochlorophyllide oxidoreductase of barley has been deduced from the nucleotide sequence of a cloned full-length cDNA. This cDNA hybridizes to a 1.7 kb RNA whose steady-state level in dark-grown seedlings is drastically reduced upon illumination. The predicted amino acid sequence (388 residues in length) includes a transit peptide of 74 amino acids whose end point has been delimited by sequencing the N-terminus of the mature protein. Expression of the cDNA in Escherichia coli leads to the synthesis of an enzymatically active precursor of the NADPH-protochlorophyllide oxidoreductase. Activity of this protein in bacterial lysates is completely dependent on the presence of NADPH and protochlorophyllide and requires light.

Amino Acid Sequence↗
PubMed1989

Light-independent NADPH-protochlorophyllide oxidoreductase activity in purified plasma membrane from the cyanobacterium Anacystis nidulans.

A light plasma membrane fraction corresponding to a buoyant density of 1.087 +/- 0.005 g/cm3 and devoid of chlorophyll was prepared and purified from Anacystis nidulans according to a recently published procedure (G.A.Peschek, V.Molitor, M.Trnka, M. Wastyn and W. Erber (1988) Methods Enzymol. 167, 437-449). Besides major amounts of carotenoids the plasma membranes contained a small but significant pool of chlorophyllide a and protochlorophyllide a as verified by room temperature and 77K spectrofluorimetry and analytical separation and identification by high performance liquid chromatography using authentic standards. Incubation of the plasma membranes in strict darkness in the presence of NADPH was accompanied by the gradual and stoichiometric replacement of protochlorophyllide by chlorophyllide, NADP+ effecting the reverse transition. The reaction was completely insensitive to illumination (5-20 w/m2 tungsten light) but abolished after heating of the membranes (90 degrees C, 5 min) or in the presence of 10 mM EGTA, and was specifically stimulated by calcium ions. Our results indicate the occurrence of light-independent NADPH:protochlorophyllide oxidoreductase activity in the plasma membrane of Anacystis nidulans.

Cell Membrane↗
PubMedDate not supplied

Characterization of three genes encoding the subunits of light-independent protochlorophyllide reductase in Chlorella protothecoides CS-41.

Light is necessary for hydrogenation of the D ring of protochlorophyllide leading to chlorophyllide formation in higher plants (light-dependent pathway), but it is not essential in phototrophic bacteria (dark pathway). Both pathways, however, occur in some algae, mosses, ferns, and gymnosperms, and each chloroplast genome of these organisms contains three genes, chlL, chlN, and chlB, encoding the three subunits of light-independent protochlorophyllide reductase (LIPOR) required for protochlorophyllide reduction in the dark. In this study, the three LIPOR genes chlL, chlN, and chlB were cloned from the chloroplast of Chlorella protothecoides CS-41 (CSIRO), which grew heterotrophically with considerable chlorophyll yield. Phylogenetic analysis of ChlL/BchL showed that C. protothecoides CS-41 and Chlorella vulgaris C-27 were closely related. Alignment of their amino acid sequences demonstrated that the conserved domains, including the ATP-binding motif and the Fe-S binding motif in the three subunits, were similar to those in nitrogenases. The three-dimensional structural model of ChlL revealed a hypothetical Fe-S center for redox control. Results from RT-PCR amplification indicated that the chlL gene in C. protothecoides contained a 951-bp intron, and the splicing catalytic core structure was similar to that of the light-regulated intron in the psbA gene of Chlamydomonas. The three genes were expressed in E. coli BL21. The sizes of ChlL, ChlN, and ChlB were estimated to be 38, 49, and 58 kDa, respectively, based on the SDS-PAGE analysis.

Amino Acid Sequence↗
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↗
PubMed2004

Origin and evolution of the light-dependent protochlorophyllide oxidoreductase (LPOR) genes.

Light-dependent NADPH-protochlorophyllide oxidoreductase (LPOR) is a nuclear-encoded chloroplast protein in green algae and higher plants which catalyzes the light-dependent reduction of protochlorophyllide to chlorophyllide. Light-dependent chlorophyll biosynthesis occurs in all oxygenic photosynthetic organisms. With the exception of angiosperms, this pathway coexists with a separate light-independent chlorophyll biosynthetic pathway, which is catalyzed by light-independent protochlorophyllide reductase (DPOR) in the dark. In contrast, the light-dependent function of chlorophyll biosynthesis is absent from anoxygenic photosynthetic bacteria. Consequently, the question is whether cyanobacteria are the ancestors of all organisms that conduct light-dependent chlorophyll biosynthesis. If so, how did photosynthetic eukaryotes acquire the homologous genes of LPOR in their nuclear genomes? The large number of complete genome sequences now available allow us to detect the evolutionary history of LPOR genes by conducting a genome-wide sequence comparison and phylogenetic analysis. Here, we show the results of a detailed phylogenetic analysis of LPOR and other functionally related enzymes in the short chain dehydrogenase/reductase (SDR) family. We propose that the LPOR gene originated in the cyanobacterial genome before the divergence of eukaryotic photosynthetic organisms. We postulated that the photosynthetic eukaryotes obtained their LPOR homologues through endosymbiotic gene transfer.

Amino Acid Sequence↗
PubMed1968

Protochlorophyllide resynthesis in dark-grown bean leaves.

The protochlorophyllide content of dark-grown bean leaves was determined at various ages. It was detectable the second day after germination and reached a maximum on about the tenth day.The resynthesis of protochlorophyllide following a single irradiation and the rate of the 685 to 673 mmu shift of the newly formed chlorophyll was examined. Leaves up to 5-days old show no lag phase in protochlorophyllide resynthesis. By the sixth day, a lag phase is evident which increases with age. The rate of the 685 to 673 mmu shift is similarly age dependent.

Journal Article↗
PubMed1969

A Short-lived Intermediate Form in the in Vivo Conversion of Protochlorophyllide 650 to Chlorophyllide 684.

When dark-grown leaves of Phaseolus vulgaris, Hordeum vulgare, Zea mays and Pisum sativum were irradiated for 3 sec at 2 degrees the first product of protochlorophyllide 650 conversion had an absorption maximum at 678 nm. This form was then converted in a dark reaction to chlorophyllide 684, the form generally observed and regarded as the in vivo product of the photoreaction. The dark conversion at 2 degrees was complete in 6 to 10 min in the various plants. The time course of the dark reaction was followed at 690 nm near the maximum of the difference spectrum for the conversion. There was a constant relationship between the initial amount of chlorophyllide 678 and the final amount of chlorophyllide 684. The rates of the dark reaction at 2 degrees varied 3-fold among the plants treated. The reaction was not first order. At 25 degrees the reaction followed at 690 nm was complete in 20 to 60 sec. Q(10)'s varied from 2.8 to 3.7 between 2 degrees and 25 degrees . Phytochrome absorbancy changes were shown to be too low to interfere with these measurements except in pea leaves. In a subsequent stage of greening newly regenerated protochlorophyllide went through the same sequence upon photoconversion. Chlorophyllide 678 probably corresponds to the product formed in vitro from the protochlorophyllide holochrome. The dark reaction appears to represent the first interaction between the photoconverted holochrome and other elements of the proplastid. The lack of this dark reaction could also account for the spectral properties of certain albino mutants.

Journal Article↗
PubMed1970

On the relationship between ribulose diphosphate carboxylase and protochlorophyllide holochrome of Phaseolus vulgaris leaves.

The relationship between ribulose diphosphate carboxylase (3-phospho-d-glycerate carboxy-lyase [dimerizing], EC 4.1.1.39, formerly known as carboxydismutase) and protochlorophyllide holochrome of etiolated Phaseolus vulgaris leaves has been studied.A procedure for partially selective extraction of the two proteins was devised using tris-HCl buffer first without and then with Triton X-100. Ribulose diphosphate carboxylase was readily extracted from etiolated bean leaves without Triton X-100, and protochlorophyllide holochrome was extracted on the addition of Triton X-100. Optimal extraction conditions for protochlorophyllide holochrome have been found to be different for tissues of different ages.

Carboxy-Lyases↗
PubMed1974

Properties of Protochlorophyllide and Chlorophyll(ide) Holochromes from Etiolated and Greening Leaves.

Protochlorophyllide and chlorophyll(ide) holochromes (Pchl-H and Chl-H) were extracted from dark-grown and greening seedlings with saponin and partly purified by ammonium sulfate fractionation. Sephadex gel filtration in the presence of saponin showed that the photoactive saponin Pchl-H from dark-grown leaves of bean (Phaseolus vulgaris L. cv. Redlands Pioneer) or pea (Pisum sativum L. cv. Greenfeast) has an apparent molecular weight of about 170,000, compared with 51,000 to 75,000 for the saponin Pchl-H from barley (Hordeum vulgare L. cv. Svalöfs Bonus). Photoconversion of saponin Pchl-H from dark-grown barley seedlings yields Chl-H with an absorption maximum at 678 nm, and with no change in apparent molecular weight. Above 0 C, a spectral shift from 678 to 672 nm follows, and a change in apparent molecular weight from about 63,000 to 29,000 is observed.Saponin Chl-H extracted from barley leaves illuminated for 15 minutes has an absorption maximum at 670 nm and an apparent molecular weight greater than 100,000. This chlorophyll holochrome has photosystem I activity and it is eluted together with the cytochromes. Saponin holochrome extracted from barley leaves returned to darkness after a light period, contains chlorophyll(ide) and protochlorophyllide complexes. Gel chromatography yields a complete separation of Chl-H (apparent molecular weight > 100,000) and photoactive Pchl-H (63,000).It is proposed that Chl-H dissociates into a chlorophyll(ide) a carrier protein complex and a photoenzyme, before the incorporation of chlorophyll into the lamellar membrane.Spectrofluorimetry on partially photoconverted preparations of saponin holochrome from barley, bean, and pea gave no indication for resonance energy transfer from protochlorophyllide to chlorophyllide. The saponin holochromes gave high polarization values, in contrast with bean holochrome extracted without the aid of detergents and bean leaves.

Journal Article↗
PubMed1977

Analysis of the subunit structure of protochlorophyllide holochrome by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

The subunit structures of protochlorophyllide holochrome (PCH) and chlorophyllide holochrome (CH) were studied by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. PCH from leaves of dark-grown (Phaseolus vulgaris var. red kidney) is a polymeric pigment-protein complex of approximately 600,000 daltons. It is composed of 12 to 14 polypeptides of 45,000 daltons, when examined prior to and immediately following photoconversion. The protochlorophyllide or chlorophyllide pigment molecules are associated with these polypeptides. Subsequent to photoconversion, the absorption maximum of newly formed chlorophyllide shifts from 678 nm to 674 nm upon standing in darkness. Following the 678 to 674 spectral shift, the chlorophyllide is associated with a polypeptide with a molecular weight of 16,000 daltons. In addition, sucrose gradient centrifugation of PCH and CH under nondenaturing conditions indicates that during the course of the dark spectroscopic shift, the 600,000 dalton CH undergoes dissociation into a small chlorophyllide protein. The dissociation of CH, the change in the molecular weight of the chlorophyllide polypeptide from 45,000 to 16,000 daltons, as well as the dark spectroscopic shift are temperature-dependent and blocked below 0 C. It was also found that each holochrome molecule of 600,000 daltons contains at least four protochlorophyllide pigment molecules.

Journal Article↗
PubMed1993

bchFNBH bacteriochlorophyll synthesis genes of Rhodobacter capsulatus and identification of the third subunit of light-independent protochlorophyllide reductase in bacteria and plants.

We present the nucleotide and deduced amino acid sequences of four contiguous bacteriochlorophyll synthesis genes from Rhodobacter capsulatus. Three of these genes code for enzymes which catalyze reactions common to the chlorophyll synthesis pathway and therefore are likely to be found in plants and cyanobacteria as well. The pigments accumulated in strains with physically mapped transposon insertion mutations are analyzed by absorbance and fluorescence spectroscopy, allowing us to assign the genes as bchF, bchN, bchB, and bchH, in that order. bchF encodes a bacteriochlorophyll alpha-specific enzyme that adds water across the 2-vinyl group. The other three genes are required for portions of the pathway that are shared with chlorophyll synthesis, and they were expected to be common to both pathways. bchN and bchB are required for protochlorophyllide reduction in the dark (along with bchL), a reaction that has been observed in all major groups of photosynthetic organisms except angiosperms, where only the light-dependent reaction has been clearly established. The purple bacterial and plant enzymes show 35% identity between the amino acids coded by bchN and chlN (gidA) and 49% identity between the amino acids coded by bchL and chlL (frxC). Furthermore, bchB is 33% identical to ORF513 from the Marchantia polymorpha chloroplast. We present arguments in favor of the probable role of ORF513 (chlB) in protochlorophyllide reduction in the dark. The further similarities of all three subunits of protochlorophyllide reductase and the three subunits of chlorin reductase in bacteriochlorophyll synthesis suggest that the two reductase systems are derived from a common ancestor.

Amino Acid Sequence↗