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PubMedDate not supplied

[Investigation of the initial steps of protochlorophyllide photoreduction in etiolated plants].

The irreversible nonfluorescent product of the protochlorophyllide photoreduction with the wide electronic band at 705 nm was discovered. The P705 product through the intermediate products with the fluorescence max at 695 nm and 687 nm turns into Chl684 678, as the result of heating in darkness. The P705, P695, P687 products quench protochlorophyllide fluorescence with great efficiency. Analysis of the intermediate product properties allow to propose a new photoreduction mechanism that includes energy transfer to the trapping center where the charge transfer is performed, nonradiative energy dissipation in the charge-transfer complex, and protonation and disproportionation of the pigment anion-radicals.

Chlorophyll↗
PubMedDate not supplied

[Energy migration in a pigmented protochlorophyllide complex].

Data are analysed concerning energy migration between protochlorophyllide and chlorophyllide formed in etiolated leaves. It is found that phototransformation of the precursor proceeds in the pigment complexes similar to the multicentric photosynthetic unit. The complexes comprise no more than 26 molecules of the active form of protochlorophyllide P650 and two molecules of minor forms of P628 and P639 on the average with mean intermolecular distances 3 nm.

Chlorophyll↗
PubMed2001

Protochlorophyllide oxidoreductase: a homology model examined by site-directed mutagenesis.

An homology model of protochlorophyllide reductase (POR) from Synechocystis sp. was constructed on a template from the tyrosine-dependent oxidoreductase family. The model showed characteristics appropriate to a globular, soluble protein and was used to generate a structure of the ternary complex of POR, nicotinamide adenine dinucleotide phosphate (NADPH), and protochlorophyllide. The POR ternary model was validated by mutagenesis experiments involving predicted coenzyme-binding residues and by chemical modification experiments. A core tryptophan residue was shown to be responsible for much of the protein's fluorescence. Both quenching of this residue by coenzyme and fluorescence resonance energy transfer (FRET) from the protein to the coenzyme allowed the binding constant of NADPH to be determined. Replacement of this residue by Tyr gave an active mutant with approximately halved fluorescence and a negligible FRET signal, consistent with the role of this residue in energy transfer to the NADPH at the active site and with the model. The mechanism of the enzyme is discussed in the context of the model and semiempirical molecular orbital calculations.

Amino Acid Sequence↗
PubMed1995

Light-enhanced gene expression of NADPH-protochlorophyllide oxidoreductase in cucumber.

The NADPH-protochlorophyllide oxidoreductase (NPR) is a well known enzyme catalyzing photoconversion of protochlorophyllide to chlorophyllide a and its expression is negatively regulated by light. We cloned a NPR cDNA from cucumber (Cucumis sativus L.). The cDNA contained an open reading frame encoding 398 amino acids and amino acid sequence showed high similarity to those of NPR from other plants. Contrasting with most other plants, the levels of the NPR mRNA increased in etiolated cotyledons of cucumber when they were illuminated with continuous light, suggesting that the expression of this NPR gene was strongly related to chlorophyll synthesis in green leaves.

Amino Acid Sequence↗
PubMed1996

Identification of three cDNA clones expressed in the leaf extension zone and with altered patterns of expression in the slender mutant of barley: a tonoplast intrinsic protein, a putative structural protein and protochlorophyllide oxidoreductase.

Three cDNA clones have been isolated on the basis of altered patterns of expression in the leaf extension zone of the developmental mutant, slender barley, compared with the wild type. mRNAs corresponding to two of the cDNAs, 7s and 8s, are increased in slender compared with normal. 7s encodes a putative gamma-TIP and is expressed throughout the elongation zone. gamma-TIPs form transmembrane channels which allow the passive transfer of water. Although expression of 7s was increased in slender leaf tissue, the increase was much less extreme than that shown by Phillips and Huttly (1994) following the application of GA to an extreme dwarf of Arabidopsis. 8s is maximally expressed in the region of early cell elongation and has 66% encoded protein identity with MFS18, a cDNA encoding a putative cell wall structural protein isolated from male flowers of maize. Both 8s and MFS18 encode small (128 amino acids) basic proteins rich in glycine, alanine, proline and serine. mRNA corresponding to the third cDNA, 24n, is present at a greatly reduced level in slender compared with normal and encodes protochlorophyllide oxidoreductase (POR). POR catalyses the conversion of protochlorophyllide into chlorophyllide. The reduced level of POR mRNA is not correlated with a similar reduction in expanded leaf blade chlorophyll levels. Western analysis identified two POR proteins present in light-grown seedlings. Whilst the larger of the proteins is present throughout most of the leaf, the smaller protein mimics the mRNA results, being both maximally present in the elongation tissue and present at a reduced level in slender. An antagonistic relationship between chlorophyll biosynthesis and extension growth is suggested.

Amino Acid Sequence↗
PubMed2006

Expression switching of three genes encoding light-independent protochlorophyllide oxidoreductase in Chlorella protothecoides.

Three chloroplast genes, chlL, chlN and chlB, encoding the light-independent protochlorophyllide oxidoreductase (LIPOR) in Chlorella protothecoides CS-41 growing either photoautotrophically, mixotrophically or heterotrophically, were all transcribed constitutively independent of illumination and presence of glucose. Steady-state amounts of all three transcripts in the light-grown cells were, however, approximately two- to three-fold greater than those in the dark-grown cells. In addition, Western blotting demonstrated that approximately the same amount of protein was present in cultures grown mixtrophically or heterotrophically both containing glucose. However, much less protein was in photoautotrophic cells. These results suggest that LIPOR activity depends on post-transcriptional and post-translational regulation. Moreover, the fact that LIPOR accumulates in the light-grown cells indicates that LIPOR, which was thought to work only in darkness, may partially account for the protochlorophyllide photoreduction in light. Therefore, LIPOR switches on both in light and in darkness.

Chlorella↗
PubMed2005

Overexpression and characterization of dark-operative protochlorophyllide reductase from Rhodobacter capsulatus.

Dark-operative protochlorophyllide oxidoreductase (DPOR) plays a crucial role in light-independent (bacterio)chlorophyll biosynthesis in most photosynthetic organisms. However, the biochemical properties of DPOR are still largely undefined. Here, we constructed an overexpression system of two separable components of DPOR, L-protein (BchL) and NB-protein (BchN-BchB), in the broad-host-range vector pJRD215 in Rhodobacter capsulatus. We established a stable DPOR assay system by mixing crude extracts from the two transconjugants under anaerobic conditions. Using this assay system, we demonstrated some basic properties of DPOR. The Km value for protochlorophyllide was 10.6 muM. Ferredoxin functioned as an electron donor to DPOR. Elution profiles in gel filtration chromatography indicated that L-protein and NB-protein are a homodimer [(BchL)(2)] and a heterotetramer [(BchN)(2)(BchB)(2)], respectively. These results provide a framework for the characterization of these components in detail, and further support a nitrogenase model of DPOR.

Base Sequence↗
PubMed2000

Identification and light-induced expression of a novel gene of NADPH-protochlorophyllide oxidoreductase isoform in Arabidopsis thaliana.

In Arabidopsis thaliana, we identified a novel gene of a NADPH-protochlorophyllide oxidoreductase (POR) isoform, which catalyzes the light-dependent protochlorophyllide a reduction in the chlorophyll (Chl) biosynthetic pathway. The deduced amino acid sequence of the novel POR isoform (PORC) showed significant identities ( approximately 75%) with the previously isolated two POR isoforms of A. thaliana. Contrasting with these POR isoforms, the PORC transcript increased in etiolated seedlings by illumination, and was dominantly expressed in immature and mature tissues. The present results demonstrated that Chl biosynthesis and chloroplast biogenesis in A. thaliana are controlled by three POR isoforms, which are differentially controlled by light and development.

Amino Acid Sequence↗
PubMed1999

The role of protein surface charge in catalytic activity and chloroplast membrane association of the pea NADPH: protochlorophyllide oxidoreductase (POR) as revealed by alanine scanning mutagenesis.

NADPH:protochlorophyllide oxidoreductase (POR) catalyzes the light-dependent reduction of protochlorophyllide (pchlide) to chlorophyllide (chlide) in the biosynthesis of chlorophyll. POR is a peripheral membrane protein that accumulates to high levels in the prolamellar bodies of vascular plant etioplasts and is present at low levels in the thylakoid membranes of developing and mature plastids. Clustered charged-to-alanine scanning mutagenesis of the pea (Pisum sativum L.) POR was carried out and the resulting mutant enzymes analyzed for their ability to catalyze pchlide photoconversion in vivo and to associate properly with thylakoid membrane preparations in vitro. Of 37 mutant enzymes examined, 5 retained wild-type levels of activity, 14 were catalytically inactive, and the remaining 18 exhibited altered levels of function. Several of the mutant enzymes showed temperature-dependent enzymatic activity, being inactive at 32 degrees C, but partially active at 24 degrees C. Mutations in predicted alpha-helical regions of the protein showed the least effect on enzyme activity, whereas mutations in predicted beta-sheet regions of the protein showed a consistent adverse affect on enzyme function. In the absence of added NADPH, neither wild-type POR nor any of the mutant PORs resisted proteolysis by thermolysin following assembly onto the thylakoid membranes. In contrast, when NADPH was present in the assay mixture, 13 of the 37 mutant PORs examined were found to be resistant to thermolysin upon treatment, suggesting that the mutations did not affect their ability to be properly attached to the thylakoid membrane. In general, the replacement of charged amino acids by alanine in the most N- and C-terminal regions of the mature protein did not significantly affect POR assembly, whereas mutations within the central core of the protein (between residues 86 and 342) were incapable of proper attachment to the thylakoid. Failure to properly associate with the thylakoid membrane in a protease resistant manner was only weakly correlated to loss of catalytic function. These studies are a first step towards defining structural determinants crucial to POR function and intraorganellar localization.

Alanine↗
PubMed2000

NADPH-protochlorophyllide oxidoreductase in cucumber is encoded by a single gene and its expression is transcriptionally enhanced by illumination.

NADPH-protochlorophyllide oxidoreductase (POR) catalyzes the photoreduction of protochlorophyllide a in the chlorophyll biosynthetic pathway. Recently, two POR genes, the expressions of which were differently regulated by light, were isolated in barley and Arabidopsis, and it is suggested that in angiosperms, the chlorophyll synthesis is controlled by the differential action of the two distinct POR isozymes. Meanwhile, we have isolated POR cDNA from cucumber, and reported that its expression was enhanced by illumination. In this study, we analyzed the gene expression and organization of cucumber POR in more detail. Northern blot analysis with 5'- or 3'-noncoding gene-specific and full length probes and RNase protection assay revealed that cucumber POR mRNA is transcribed from a single gene. Run-on assay revealed that the transcript level of isolated nuclei from illuminated cotyledons was 6-fold that of dark control, indicating that the light-enhanced expression of cucumber POR is caused by transcriptional activation. Using degenerated primers and the genomic DNA of cucumber as template, PCR showed the presence of only one type of fragment encoding cucumber POR. The PCR product was hybridized with the genomic Southern blot from cucumber and only one band was detected under low stringency conditions. From these results, we conclude that there is a single POR gene in cucumber and PORs are organized by different gene families among higher plants, and in cucumber the single POR may play the roles of two POR genes, which are present in other higher plants such as Arabidopsis and barley.

Journal Article↗
PubMed2002

Identification of two differentially regulated isoforms of protochlorophyllide oxidoreductase (POR) from tobacco revealed a wide variety of light- and development-dependent regulations of POR gene expression among angiosperms.

NADPH-protochlorophyllide oxidoreductase (POR) catalyzes the light-dependent reduction of protochlorophyllide a in the chlorophyll biosynthetic pathway. Here, we identified two distinct POR cDNAs from tobacco. Both POR isoforms are encoded by a respective single copy gene in tobacco genome. The overall deduced amino acid sequences of two tobacco cDNAs, designated here POR1 and POR2, displayed significant identities ( approximately 75%), but showed different patterns of light and developmental regulation. In contrast to the previously isolated POR isoforms of Arabidopsis thaliana and barley, the expression of both tobacco POR isoforms were not negatively regulated by light and persisted in matured green tissues. Furthermore, the expression of both genes appeared to be regulated by a diurnal regulation. These results show a wide variety of light- and development-dependent regulations of POR gene expression among angiosperms. Furthermore, phylogenetic analysis including tobacco revealed that POR gene family is differentially represented by angiosperms, most of which is probably caused by independent gene duplication in individual plant. Present results imply a modification of the previous concept that chlorophyll biosynthesis and chloroplast differentiation in angiosperms are ubiquitously controlled by unique functions of two POR isoforms.

Journal Article↗
PubMed2004

Novel Insights into the Enzymology, Regulation and Physiological Functions of Light-dependent Protochlorophyllide Oxidoreductase in Angiosperms.

The reduction of protochlorophyllide (Pchlide) is a key regulatory step in the biosynthesis of chlorophyll in phototrophic organisms. Two distinct enzymes catalyze this reduction; a light-dependent NADPH:protochlorophyllide oxidoreductase (POR) and light-independent Pchlide reductase (DPOR). Both enzymes are widely distributed among phototrophic organisms with the exception that only POR is found in angiosperms and only DPOR in anoxygenic photosynthetic bacteria. Consequently, angiosperms become etiolated in the absence of light, since the reduction of Pchlide in angiosperms is solely dependent on POR. In eukaryotic phototrophs, POR is a nuclear-encoded single polypeptide and post-translationally imported into plastids. POR possesses unique features, its light-dependent catalytic activity, accumulation in plastids of dark-grown angiosperms (etioplasts) via binding to its substrate, Pchlide, and cofactor, NADPH, resulting in the formation of prolamellar bodies (PLBs), and rapid degradation after catalysis under subsequent illumination. During the last decade, considerable progress has been made in the study of the gene organization, catalytic mechanism, membrane association, regulation of the gene expression, and physiological function of POR. In this review, we provide a brief overview of DPOR and then summarize the current state of knowledge on the biochemistry and molecular biology of POR mainly in angiosperms. The physiological and evolutional implications of POR are also discussed.

Journal Article↗
PubMed2005

Kinetic characterisation of the light-driven protochlorophyllide oxidoreductase (POR) from Thermosynechococcus elongatus.

The light-driven enzyme NADPH:protochlorophyllide oxidoreductase (POR) catalyses the reduction of the C17-C18 double bond of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), which is a key regulatory step in the chlorophyll biosynthesis pathway. POR from the thermophilic cyanobacterium Thermosynechococcus elongatus is an attractive system for following the reaction and in the present work we have carried out a detailed steady state kinetic characterisation of this enzyme. The thermophilic POR was shown to have maximal activity at approximately 50 degrees C, which is similar to the growth temperature of the organism. The V(max) was calculated to be 0.53 microM min(-1) and the K(m) values for NADPH and Pchlide were 0.013 microM and 1.8 microM, respectively. The binding properties for both substrates as well as the NADP(+) product have been analysed by using fluorescence emission measurements, which have allowed the dissociation constants for binding to be calculated. These results represent the first steady state kinetic characterisation of a thermophilic version of POR.

Catalysis↗
PubMed1981

Covalent labelling of the NADPH: protochlorophyllide oxidoreductase from etioplast membranes with [3H]N-phenylmaleimide.

[3H]N-Phenylmaleimide has been used to covalently label in a specific manner the substrate-protected thiol groups of the enzyme protochlorophyllide reductase. In membrane preparations from oat (Avena sativa) and runner-bean (Phaseolus vulgaris) seedlings, two related peptides of mol.wts. 35000/37000 and 34000/35000 respectively and showing properties expected of the reductase have been identified, whereas the same technique with barley (Hordeum vulgare) extracts resulted in labelling a single peptide of mol.wt. 38000. Evidence is presented that both NADPH and protochlorophyllide are required for protection of the essential thiol groups on the reductase in oat extracts, a situation favouring a ternary complex as the structure for the photoactive enzyme--substrates intermediate.

Chloroplasts↗
PubMed1995

Protochlorophyllide reductase in photosynthetic prokaryotes and its role in chlorophyll synthesis.

1. DNA sequences hybridizing with the wheat protochlorophyllide reductase gene have been detected during genomic Southern blots of various cyanobacterial DNA samples. No such hybridization was observed with DNA from photosynthetic bacteria. 2. A fragment amplified from Phormidium laminosum DNA has been characterized and shown to be 73% similar to the corresponding wheat sequence. At the protein level the similarity is 91%. When used as a probe for Southern blotting the Phormidium DNA fragment confirmed the authenticity of some of the original signals obtained with the wheat probe. 3. Peptides of molecular mass 36, 30 and 60 kDa are immunodetected by a wheat reductase antibody during Western blotting of Phormidium preparations. These are purported to correspond to the cyanobacterial mature reductase protein, a stable proteolytic fragment and soluble dimeric forms of the latter respectively. 4. Adaptation of Phormidium to growth in red light (delta > 670 nm) or darkness led to no significant changes in the total level of immunodetected peptides or protochlorophyllide within the cells. 5. The specific activity of the reductase in Phormidium membranes has been tentatively estimated as 0.5 unit/mg of protein, a value comparable with that found in preparations from mature chloroplasts of higher plants.

Amino Acid Sequence↗
PubMed2002

Site-directed mutagenesis of Tyr-189 and Lys-193 in NADPH: protochlorophyllide oxidoreductase from Synechocystis.

NADPH:protochlorophyllide oxidoreductase (POR) catalyses the light-dependent reduction of protochlorophyllide to chlorophyllide, a key regulatory reaction in the chlorophyll biosynthetic pathway. Sequence comparisons have revealed that POR is a member of the short-chain alcohol dehydrogenase family of enzymes. A tyrosine and a lysine residue are conserved throughout all members of this family, and are proposed to be within the active site. This present study describes how site-directed mutagenesis has been used to change Tyr-189 to Phe and Lys-193 to Arg in the Synechocystis POR enzyme. The mutant enzymes were produced with a His tag in Escherichia coli and subsequently purified on a Ni(2+)-affinity column. The two mutations resulted in inactive enzymes, indicating that both residues are crucial for activity. The K(d) value for NADPH binding to the K193R mutant was significantly higher than for the wild-type enzyme, suggesting that the affinity for NADPH has also been reduced.

Amino Acid Substitution↗
PubMed1996

Distinct roles for light-dependent NADPH:protochlorophyllide oxidoreductases (POR) A and B during greening in higher plants.

Light-dependent NADPH:protochlorophyllide oxidoreductase (POR), a nuclear-encoded plastid-localized enzyme, catalyzes the photoreduction of protochlorophyllide (Pchlide) to chlorophyllide in higher plants, algae and cyanobacteria. Angiosperms require light for chlorophyll (Chl) biosynthesis and have recently been shown to contain two POR-encoding genes, PorA and PorB, that are differentially regulated by light and developmental state. PorA expression rapidly becomes undetectable after illumination of etiolated seedlings, whereas PorB expression persists throughout greening and in adult plants. In order to study the in vivo functions of Arabidopsis POR A and POR B we have abolished the expression of PorA through the use of the phytochrome A-mediated far-red high irradiance response. Wild-type seedlings grown in continuous far-red light (cFR) display the morphology of white light (WL)-grown seedlings, but contain only traces of Chl and do not green upon transfer to WL. This cFR-induced greening defect correlates with the absence of PorA mRNA, the putative POR A protein, phototransformable Pchlide-F655, and with strongly reduced POR enzymatic activity in plant extracts. In contrast, a cFR-grown phyA mutant expresses the PorA gene, accumulates Chl and visibly greens in WL. Furthermore, constitutive overexpression of POR A in cFR-grown transgenic Arabidopsis wild-type seedlings restores Chl accumulation and WL-induced greening. These data demonstrate that POR A is required for greening and that the availability of POR A limits Chl accumulation during growth in cFR. POR B apparently provides a means to sustain light-dependent Chl biosynthesis in fully greened, mature plants in the absence of phototransformable Pchlide-F655.

Arabidopsis↗
PubMed2002

Enzymology below 200 K: the kinetics and thermodynamics of the photochemistry catalyzed by protochlorophyllide oxidoreductase.

The chlorophyll biosynthesis enzyme protochlorophyllide reductase (POR) catalyzes the light-dependent reduction of protochlorophyllide (Pchlide) into chlorophyllide in the presence of NADPH. As POR is light-dependent, catalysis can be initiated by illumination of the enzyme-substrate complex at low temperatures, making it an attractive model for studying aspects of biological proton and hydride transfers. The early stages in the photoreduction, involving Pchlide binding and an initial photochemical reaction, have been studied in vitro by using low-temperature fluorescence and absorbance measurements. Formation of the ternary POR-NADPH-Pchlide complex produces red shifts in the fluorescence and absorbance maxima of Pchlide, allowing the dissociation constant for Pchlide binding to be measured. We demonstrate that the product of an initial photochemical reaction, which can occur below 200 K, is a nonfluorescent intermediate with a broad absorbance band at 696 nm (A696) that is suggested to represent an ion radical complex. The temperature dependence of the rate of A696 formation has allowed the activation energy for the photochemical step to be calculated and has shown that POR catalysis can proceed at much lower temperatures than previously thought. Calculations of differences in free energy between various reaction intermediates have been calculated; these, together with the quantum efficiency for Pchlide conversion, suggest a quantitative model for the thermodynamics of the light-driven step of Pchlide reduction.

Binding Sites↗