The Experts below are selected from a list of 204 Experts worldwide ranked by ideXlab platform
Christer Sundqvist - One of the best experts on this subject based on the ideXlab platform.
-
ADP/ATP and protein phosphorylation dependence of phototransformable Protochlorophyllide in isolated etioplast membranes
Photosynthesis Research, 2000Co-Authors: Sabina Kovacheva, Margareta Ryberg, Christer SundqvistAbstract:The effects of modulated ADP/ATP and NADPH/NADP^+ ratios, and of protein kinase inhibitors, on the in vitro reformation of phototransformable Protochlorophyllide, i.e. the aggregated ternary complexes between NADPH, Protochlorophyllide, and NADPH-Protochlorophyllide oxidoreductase (POR, EC 1.3.1.33), in etioplast membranes isolated from dark-grown wheat ( Triticum aestivum ) were investigated. Low temperature fluorescence emission spectra (–196 °C) were used to determine the state of the pigments. The presence of spectral intermediates of Protochlorophyllide and the reformation of phototransformable Protochlorophyllide were reduced at high ATP, but favoured by high ADP. Increased ADP level partly prevented the chlorophyllide blue-shift. The protein kinase inhibitor K252a prevented reformation of phototransformable Protochlorophyllide without showing any effect on the chlorophyllide blue-shift. Addition of NADPH did not overcome the inhibition. The results indicate that protein phosphorylation plays a role in the conversion of the non-phototransformable Protochlorophyllide to POR-associated phototransformable Protochlorophyllide. The possible presence of a plastid ADP-dependent kinase, the activity of which favours the formation of PLBs, is discussed. Reversible protein phosphorylation is suggested as a regulatory mechanism in the prolamellar body formation and its light-dependent dispersal by affecting the membrane association of POR. By the presence of a high concentration of phototransformable Protochlorophyllide, prolamellar bodies can act as light sensors for plastid development. The modulation of plastid protein kinase and protein phosphatase activities by the NADPH/NADP^+ ratio is suggested.
-
Protochlorophyllide and Por in the Lip1 Mutant of Pea
The Chloroplast: From Molecular Biology to Biotechnology, 1999Co-Authors: M. Seyyedi, Michael P. Timko, Christer SundqvistAbstract:The prolamellar bodies (PLBs) have a high content of the Protochlorophyllide (Pchlide) reducing enzyme NADPH-Protochlorophyllide oxidoreductase (POR). The genes for two different POR enzymes (PORA and PORB) have been isolated from barely and Arabidopsis [1, 2]. PORA is present mostly in dark-grown plants while PORB is more persistent and is suggested to be the main chlorophyll forming enzyme [3]. However, in pea only one type of POR seems to be present [4].
-
the two spectroscopically different short wavelength Protochlorophyllide forms in pea epicotyls are both monomeric
Biochimica et Biophysica Acta, 1998Co-Authors: Béla Böddi, Katalin Kispetik, Andras D Kaposi, Judit Fidy, Christer SundqvistAbstract:Abstract The spectral properties of the Protochlorophyllide forms in the epicotyls of dark-grown pea seedlings have been studied in a temperature range, from 10 to 293 K with conventional fluorescence emission and excitation spectroscopy as well as by fluorescence line narrowing (FLN) at cryogenic temperatures. The conventional fluorescence techniques at lower temperatures revealed separate bands at 628, 634–636, 644 and 655 nm. At room temperature (293 K) the 628 and 634–636 nm emission bands strongly overlapped and the band shape was almost independent of the excitation wavelength. Under FLN conditions, vibronically resolved fluorescence spectra could be measured for the 628 and 634–636 nm bands. The high resolution of this technique excluded the excitonic nature of respective excited states and made it possible to determine the pure electronic (0,0) range of the spectra of the two components. Thus it was concluded that the 628 and 634–636 nm (0,0) emission bands originate from two monomeric forms of Protochlorophyllide and the spectral difference is interpreted as a consequence of environmental effects of the surrounding matrix. On the basis of earlier results and the data presented here, a model is discussed in which the 636 nm form is considered as an enzyme-bound Protochlorophyllide and the 628 nm form as a Protochlorophyllide pool from which the substrate is replaced when the epicotyl is illuminated with continuous light.
-
Protochlorophyllide transformations and chlorophyll accumulation in epicotyls of pea (Pisum sativum)
Physiologia Plantarum, 1996Co-Authors: Béla Böddi, Margareta Ryberg, Ivar Evertsson, Christer SundqvistAbstract:Low-temperature fluorescence emission spectra of epicotyls of 6.5-day-old dark-grown seedlings of pea (Pisum sativum L.) showed the dominance of short-wavelength protoch lorophyllide forms with emission maxima at 629 and 636 nm, respectively. The presence of long-wavelength Protochlorophyllide with emission maxima around 650 nm was just detectable. Accordingly, irradiation with millisecond flashes gave a minute formation of chlorophyllide. The chlorophyll(ide) formation varied along the epicotyl. Irradiation with continuous light for 1.5 h resulted in an evident accumulation of chlorophyll(ide) in the upper part of the epicotyl. Only small amounts accumulated in the middle section. The conversion of Protochlorophyllide to chlorophyllide was temperature dependent and almost arrested at 0°C. The chlorophyll(ide) formed had one dominating fluorescence peak at 681 nm. Irradiation for 24 h gave almost 100 times more chlorophyll in the upper part of the epicotyl than in the lower part. Electron micrographs from the upper part of the epicotyl irradiated for 6 h showed plastids with several developing thylakoids, while the plastids in the lower part of the epicotyl had only a few thylakoids. The dominance of short-wavelength Protochlorophyllide forms indicated the presence of Protochlorophyllide not bound to the active site of NADPH-Protochlorophyllide oxidoreductase (EC 1.3.1.33). The inability of the short-wavelength form to transform into chlorophyllide with flash light denotes a dislocation from the active site. The time and temperature dependence of the chlorophyll(ide) formation in continuous light indicates that a relocation is required of the short-wavelength Protochlorophyllide before chlorophyllide formation can occur.
-
Photoreduction of zinc protopheophorbide b with NADPH-Protochlorophyllide oxidoreductase from etiolated wheat (Triticum aestivum L.).
European journal of biochemistry, 1995Co-Authors: Siegrid Schoch, Michael Helfrich, Wolfhart Rüdiger, Christer Sundqvist, Bengt Wiktorsson, Margareta RybergAbstract:A preparation of prolamellar bodies from wheat etioplasts was used as a source for NADPH-Protochlorophyllide oxidoreductase (pchlide reductase). The enzyme was solubilized with Triton X-100 after reduction of the endogenous photoconvertible Protochlorophyllide a to chlorophyllide a by saturating illumination. Protochlorophylls a and b, Protochlorophyllide a and zinc protopheophorbide b were added to the soluble enzyme preparation to determine if they were reduced in the dark or in the light. None of the compounds were reduced (with NADPH) in the dark; however, light-dependent reduction was demonstrated with Protochlorophyllide a and zinc protopheophorbide b. The yield was approximately 50% for both substrates. Photoreduction did not occur with the esterified protochlorophylls a and b. Photoreduction of zinc protopheophorbide b, the zinc analogue of Protochlorophyllide b, is the first demonstration of the reduction of a chlorophyll-b-related compound by pchlide reductase.
Béla Böddi - One of the best experts on this subject based on the ideXlab platform.
-
distinct uv a or uv b irradiation induces Protochlorophyllide photoreduction and bleaching in dark grown pea pisum sativum l epicotyls
Photosynthesis Research, 2019Co-Authors: Annamaria Kosa, Anna Laura Erdei, Béla BöddiAbstract:The effects of distinct UV-A and UV-B radiations were studied on etiolated pea (Pisum sativum L.) epicotyls. Emission spectra of the native protochlorophyll and Protochlorophyllide forms were measured when epicotyls were excited with 360 or 300 nm light. The UV-A (360 nm) excited mainly the non-enzyme-bound monomers of protochlorophyll and Protochlorophyllide and the UV-B (300 nm) excited preferentially the flash-photoactive Protochlorophyllide complexes. These latter complexes converted into short- and long-wavelength chlorophyllide forms at 10-s illumination with both wavelength irradiations. As the spectral changes were very small, the effects of longer illumination periods were studied. Room temperature fluorescence emission spectra were measured from the same epicotyl spots before and after irradiation with various wavelengths between 280 and 360 nm for 15 min and the “illuminated” minus “dark” difference spectra were calculated. Both the UV-A and the UV-B irradiations caused photoreduction of Protochlorophyllide into chlorophyllide. At 10 µmol photons m−2 s−1, the photoreduction rates were similar, however, at 60 µmol photons m−2 s−1, the UV-B irradiation was more effective in inducing chlorophyllide formation than the UV-A. The action spectra of Protochlorophyllide plus protochlorophyll loss and chlorophyllide production showed that the radiation around 290 nm was the most effective in provoking Protochlorophyllide photoreduction and the UV light above 320 nm caused strong bleaching. These results show that the effect of the UV radiation should be considered when discussing the Protochlorophyllide–chlorophyllide photoreduction during germination and as a part of the regeneration of the photosynthetic apparatus proceeding in the daily run of photosynthesis.
-
the two spectroscopically different short wavelength Protochlorophyllide forms in pea epicotyls are both monomeric
Biochimica et Biophysica Acta, 1998Co-Authors: Béla Böddi, Katalin Kispetik, Andras D Kaposi, Judit Fidy, Christer SundqvistAbstract:Abstract The spectral properties of the Protochlorophyllide forms in the epicotyls of dark-grown pea seedlings have been studied in a temperature range, from 10 to 293 K with conventional fluorescence emission and excitation spectroscopy as well as by fluorescence line narrowing (FLN) at cryogenic temperatures. The conventional fluorescence techniques at lower temperatures revealed separate bands at 628, 634–636, 644 and 655 nm. At room temperature (293 K) the 628 and 634–636 nm emission bands strongly overlapped and the band shape was almost independent of the excitation wavelength. Under FLN conditions, vibronically resolved fluorescence spectra could be measured for the 628 and 634–636 nm bands. The high resolution of this technique excluded the excitonic nature of respective excited states and made it possible to determine the pure electronic (0,0) range of the spectra of the two components. Thus it was concluded that the 628 and 634–636 nm (0,0) emission bands originate from two monomeric forms of Protochlorophyllide and the spectral difference is interpreted as a consequence of environmental effects of the surrounding matrix. On the basis of earlier results and the data presented here, a model is discussed in which the 636 nm form is considered as an enzyme-bound Protochlorophyllide and the 628 nm form as a Protochlorophyllide pool from which the substrate is replaced when the epicotyl is illuminated with continuous light.
-
Protochlorophyllide transformations and chlorophyll accumulation in epicotyls of pea (Pisum sativum)
Physiologia Plantarum, 1996Co-Authors: Béla Böddi, Margareta Ryberg, Ivar Evertsson, Christer SundqvistAbstract:Low-temperature fluorescence emission spectra of epicotyls of 6.5-day-old dark-grown seedlings of pea (Pisum sativum L.) showed the dominance of short-wavelength protoch lorophyllide forms with emission maxima at 629 and 636 nm, respectively. The presence of long-wavelength Protochlorophyllide with emission maxima around 650 nm was just detectable. Accordingly, irradiation with millisecond flashes gave a minute formation of chlorophyllide. The chlorophyll(ide) formation varied along the epicotyl. Irradiation with continuous light for 1.5 h resulted in an evident accumulation of chlorophyll(ide) in the upper part of the epicotyl. Only small amounts accumulated in the middle section. The conversion of Protochlorophyllide to chlorophyllide was temperature dependent and almost arrested at 0°C. The chlorophyll(ide) formed had one dominating fluorescence peak at 681 nm. Irradiation for 24 h gave almost 100 times more chlorophyll in the upper part of the epicotyl than in the lower part. Electron micrographs from the upper part of the epicotyl irradiated for 6 h showed plastids with several developing thylakoids, while the plastids in the lower part of the epicotyl had only a few thylakoids. The dominance of short-wavelength Protochlorophyllide forms indicated the presence of Protochlorophyllide not bound to the active site of NADPH-Protochlorophyllide oxidoreductase (EC 1.3.1.33). The inability of the short-wavelength form to transform into chlorophyllide with flash light denotes a dislocation from the active site. The time and temperature dependence of the chlorophyll(ide) formation in continuous light indicates that a relocation is required of the short-wavelength Protochlorophyllide before chlorophyllide formation can occur.
-
Short-wavelength Protochlorophyllide forms and their phototransformation in epicotyl of pea (Pisum sativum)
1995Co-Authors: Béla Böddi, Birgitta Mc Ewen, Margaretha Ryberg, Christer SundqvistAbstract:Short-wavelength Protochlorophyllide forms and their phototransformation in epicotyl of pea (Pisum sativum)
Margareta Ryberg - One of the best experts on this subject based on the ideXlab platform.
-
ADP/ATP and protein phosphorylation dependence of phototransformable Protochlorophyllide in isolated etioplast membranes
Photosynthesis Research, 2000Co-Authors: Sabina Kovacheva, Margareta Ryberg, Christer SundqvistAbstract:The effects of modulated ADP/ATP and NADPH/NADP^+ ratios, and of protein kinase inhibitors, on the in vitro reformation of phototransformable Protochlorophyllide, i.e. the aggregated ternary complexes between NADPH, Protochlorophyllide, and NADPH-Protochlorophyllide oxidoreductase (POR, EC 1.3.1.33), in etioplast membranes isolated from dark-grown wheat ( Triticum aestivum ) were investigated. Low temperature fluorescence emission spectra (–196 °C) were used to determine the state of the pigments. The presence of spectral intermediates of Protochlorophyllide and the reformation of phototransformable Protochlorophyllide were reduced at high ATP, but favoured by high ADP. Increased ADP level partly prevented the chlorophyllide blue-shift. The protein kinase inhibitor K252a prevented reformation of phototransformable Protochlorophyllide without showing any effect on the chlorophyllide blue-shift. Addition of NADPH did not overcome the inhibition. The results indicate that protein phosphorylation plays a role in the conversion of the non-phototransformable Protochlorophyllide to POR-associated phototransformable Protochlorophyllide. The possible presence of a plastid ADP-dependent kinase, the activity of which favours the formation of PLBs, is discussed. Reversible protein phosphorylation is suggested as a regulatory mechanism in the prolamellar body formation and its light-dependent dispersal by affecting the membrane association of POR. By the presence of a high concentration of phototransformable Protochlorophyllide, prolamellar bodies can act as light sensors for plastid development. The modulation of plastid protein kinase and protein phosphatase activities by the NADPH/NADP^+ ratio is suggested.
-
Protochlorophyllide transformations and chlorophyll accumulation in epicotyls of pea (Pisum sativum)
Physiologia Plantarum, 1996Co-Authors: Béla Böddi, Margareta Ryberg, Ivar Evertsson, Christer SundqvistAbstract:Low-temperature fluorescence emission spectra of epicotyls of 6.5-day-old dark-grown seedlings of pea (Pisum sativum L.) showed the dominance of short-wavelength protoch lorophyllide forms with emission maxima at 629 and 636 nm, respectively. The presence of long-wavelength Protochlorophyllide with emission maxima around 650 nm was just detectable. Accordingly, irradiation with millisecond flashes gave a minute formation of chlorophyllide. The chlorophyll(ide) formation varied along the epicotyl. Irradiation with continuous light for 1.5 h resulted in an evident accumulation of chlorophyll(ide) in the upper part of the epicotyl. Only small amounts accumulated in the middle section. The conversion of Protochlorophyllide to chlorophyllide was temperature dependent and almost arrested at 0°C. The chlorophyll(ide) formed had one dominating fluorescence peak at 681 nm. Irradiation for 24 h gave almost 100 times more chlorophyll in the upper part of the epicotyl than in the lower part. Electron micrographs from the upper part of the epicotyl irradiated for 6 h showed plastids with several developing thylakoids, while the plastids in the lower part of the epicotyl had only a few thylakoids. The dominance of short-wavelength Protochlorophyllide forms indicated the presence of Protochlorophyllide not bound to the active site of NADPH-Protochlorophyllide oxidoreductase (EC 1.3.1.33). The inability of the short-wavelength form to transform into chlorophyllide with flash light denotes a dislocation from the active site. The time and temperature dependence of the chlorophyll(ide) formation in continuous light indicates that a relocation is required of the short-wavelength Protochlorophyllide before chlorophyllide formation can occur.
-
Photoreduction of zinc protopheophorbide b with NADPH-Protochlorophyllide oxidoreductase from etiolated wheat (Triticum aestivum L.).
European journal of biochemistry, 1995Co-Authors: Siegrid Schoch, Michael Helfrich, Wolfhart Rüdiger, Christer Sundqvist, Bengt Wiktorsson, Margareta RybergAbstract:A preparation of prolamellar bodies from wheat etioplasts was used as a source for NADPH-Protochlorophyllide oxidoreductase (pchlide reductase). The enzyme was solubilized with Triton X-100 after reduction of the endogenous photoconvertible Protochlorophyllide a to chlorophyllide a by saturating illumination. Protochlorophylls a and b, Protochlorophyllide a and zinc protopheophorbide b were added to the soluble enzyme preparation to determine if they were reduced in the dark or in the light. None of the compounds were reduced (with NADPH) in the dark; however, light-dependent reduction was demonstrated with Protochlorophyllide a and zinc protopheophorbide b. The yield was approximately 50% for both substrates. Photoreduction did not occur with the esterified protochlorophylls a and b. Photoreduction of zinc protopheophorbide b, the zinc analogue of Protochlorophyllide b, is the first demonstration of the reduction of a chlorophyll-b-related compound by pchlide reductase.
-
THE FORMATION OF A SHORT-WAVELENGTH CHLOROPHYLLIDE FORM AT PARTIAL PHOTOTRANSFORMATION OF Protochlorophyllide IN ETIOPLAST INNER MEMBRANES
Photochemistry and Photobiology, 1991Co-Authors: Bela Böuddi, Margareta Ryberg, Christer SundqvistAbstract:— Partial phototransformation caused the formation and successive accumulation of spectral forms of chlorophyllide in etioplast inner membrane fractions from wheat (Triticum aestivum L. cv. Walde), Low-temperature fluorescence emission and circular dichroism spectra showed the formation of two chlorophyllide forms with emission maxima at 683 and 694 nm, respectively. The light dependent accumulation of the two forms differed in prolamellar body (PLB) and prothylakoid (PT) fractions. The 694 nm form was preferentially found in PLB fractions which before irradiation were characterized by a 657 nm emitting Protochlorophyllide form and a regular PLB structure. The 683 nm form accumulated to a higher extent in PT fractions which before irradiation contained mainly shortwavelength Protochlorophyllide forms. The results indicate that at least two photoactive Protochlorophyllide forms must be considered. The major part of Protochlorophyllide, with a fluoresence emission maximum at 657 nm, is transformed to the 694 nm emitting chlorophyllide first at high light doses. A minor part, revealed only after Gaussian resolution, has a fluorescence emission maximum at 645 nm and was transformed by low light doses to chlorophyllide emitting at 683 nm.
Michael P. Timko - One of the best experts on this subject based on the ideXlab platform.
-
NADPH: Protochlorophyllide Oxidoreductase-Structure, Catalytic Function, and Role in Prolamellar Body Formation and Morphogenesis
2013Co-Authors: Michael P. TimkoAbstract:The biosynthesis of chlorophyll is a critical biochemical step in the development of photosynthetic vascular plants and green algae. From photosynthetic bacteria (cyanobacteria) to algae, non-vascular plants, gymnosperms and vascular plants, mechanisms have evolved for Protochlorophyllide reduction a key step in chlorophyll synthesis. Protochlorophyllide reduction is carried out by both a light-dependent (POR) and light-independent (LIPOR) mechanisms. NADPH: Protochlorophyllide oxidoreductase (EC 1.3.1.33, abbreviated POR) catalyzes the light-dependent reduction of Protochlorophyllide (PChlide) to chlorophyllide (Chlide). In contrast, a light-independent Protochlorophyllide reductase (LIPOR) involves three plastid gene products (chlL, chlN, and chlB) and several nuclear factors. Our work focused on characterization of both the POR and LIPOR catalyzed processes.
-
Protochlorophyllide and Por in the Lip1 Mutant of Pea
The Chloroplast: From Molecular Biology to Biotechnology, 1999Co-Authors: M. Seyyedi, Michael P. Timko, Christer SundqvistAbstract:The prolamellar bodies (PLBs) have a high content of the Protochlorophyllide (Pchlide) reducing enzyme NADPH-Protochlorophyllide oxidoreductase (POR). The genes for two different POR enzymes (PORA and PORB) have been isolated from barely and Arabidopsis [1, 2]. PORA is present mostly in dark-grown plants while PORB is more persistent and is suggested to be the main chlorophyll forming enzyme [3]. However, in pea only one type of POR seems to be present [4].
-
Protochlorophyllide photoreduction
Photosynthesis Research, 1998Co-Authors: Nikolai Lebedev, Michael P. TimkoAbstract:Chlorophylls play a fundamental role in the absorption of light energy and its conversion into chemical energy for use by all organisms living on the Earth. The formation of these compounds occurs by a complex series of reactions carried out throughout the lifetime of the plant. One step of this reaction series, the conversion of Protochlorophyllide into chlorophyllide, is unique in its requirement for light. The reduction of Protochlorophyllide to chlorophyllide is catalyzed by the nuclear-encoded enzyme NADPH: Protochlorophyllide oxidoreductase (POR). Recently, genes encoding two different POR proteins, designated PORa and PORb, have been identified in vascular plants. Despite a high degree of amino acid sequence similarity, the roles played by these different gene products appear to be quite distinct during photomorphogenesis. While PORb is present and active throughout the life of the plant, PORa appears to be present and functional only in the first few hours of greening following the onset of illumination of etiolated (or dark-adapted) plants. Consistent with this, analysis of gene expression patterns showed that the transcription of the PORa and PORb encoding genes are controlled differently by light and plant developmental stage. PORa transcription is negatively photoregulated by light. Phytochrome, mainly Phytochrome a, is responsible for light control of the gene transcription. Import of PORa but not PORb into plastids also appears to be differentially regulated, with pPORa translocation across the chloroplast envelope being controlled by substrate (Protochlorophyllide) availability. In etiolated plants, POR forms large aggregates located in tube-like structures termed, ‘prolamellar bodies’. Some amount of POR was detected in prothylakoids. In light-adapted vascular plants, green algae and cyanobacteria, POR and Protochlorophyllide were detected in chloroplast envelope and stroma membranes. Photoactive enzyme complexes are identified mainly in stroma membranes near polyribosomes. The unique feature of POR is that it is a photo-enzyme whose catalytic activity depends on light. It also requires NADPH and undergoes conformational changes near Cys groups in the course of catalytic activity. The first step of the reaction is photo-induced electron transfer to Protochlorophyllide that leads to the formation of a nonfluorescent ion-radical. This step is followed by hydrogen transfer from NADPH. Degradation of PORa is specifically controlled and performed by the nuclear encoded proteases. The mechanism of the light activation of these proteases expression is unknown, but they appear only after onset of illumination. Their activity also depends on the susceptibility of POR after its conformational change followed by Protochlorophyllide photoreduction. Analysis of chlorophyll biosynthesis in chloroplasts of light-adapted plants revealed a new mechanism of Protochlorophyllide photo-reduction. The reaction differs from that previously observed in etiolated plants by the initial photoactive Protochlorophyllide form, intermediate steps and the final product. The rates of chlorophyll accumulation through these two reactions also differ. The same reaction was also detected in etiolated plants in parallel to the main reaction of Protochlorophyllide photoreduction. Study of different photosynthetic organisms reveals the presence of POR in all of them including higher plants, green algae and cyanobacteria, which possess for light-independent chlorophyll synthesis, except for photosynthetic bacteria. Among those, PORa is present only in angiosperms, which lost the ability to synthesize chlorophyll in the dark. Sequence similarity reveals the evolutionary origin of POR from short-chain alcohol dehydrogenases.
-
The pc-1 phenotype of Chlamydomonas reinhardtii results from a deletion mutation in the nuclear gene for NADPH:Protochlorophyllide oxidoreductase
Plant Molecular Biology, 1996Co-Authors: Michael P. TimkoAbstract:The pc-1 mutant of Chlamydomonas reinhardtii has been shown to be incapable of Protochlorophyllide photoconversion in vivo and is thought to be defective in light-dependent NADPH:Protochlorophyllide oxidoreductase activity. We have isolated and characterized the nuclear genes encoding this enzyme from wild-type and pc-1 mutant Chlamydomonas cells. The wild-type CRlpcr-1 gene encodes a 397 amino acid polypeptide of which the N-terminal 57 residues comprise the chloroplast transit sequence. The Chlamydomonas Protochlorophyllide reductase has 66–70% identity (79–82% similarity) to the higher plant enzymes. Transcripts encoding Protochlorophyllide reductase are abundant in dark-grown wild-type cells, but absent or at very low levels in cells grown in the light. Similarily, immunoreactive Protochlorophyllide reductase protein is also present to a greater extent in dark-versus light-grown wild-type cells. Both pc-1 and pc-1 y-7 cells lack CRlpcr-1 mRNA and the major (36 kDa) immunodetectable form of Protochlorophyllide reductase consistent with their inability to photoreduce Protochlorophyllide. DNA sequence analysis revealed that the lpcr gene in pc-1 y-7 cells contains a two-nucleotide deletion within the fourth and fifth codons of the Protochlorophyllide reductase precursor that causes a shift in the reading frame and results in premature termination of translation. The absence of Protochlorophyllide reductase message in pc-1 and pc-1 y-7 cells is likely the consequence of this frameshift mutation in the lpcr gene. Introduction of the CRlpcr-1 gene into pc-1 y-7 cells by nuclear transformation was sufficient to restore the wild-type phenotype. Transformants contained both Protochlorophyllide reductase mRNA and immunodetectable enzyme protein. These studies demonstrate that pc-1 was in fact a defect in Protochlorophyllide reductase activity and provide the first in vivo molecular evidence that the lpcr gene product is essential for light-dependent Protochlorophyllide reduction.
-
a light dependent complementation system for analysis of nadph Protochlorophyllide oxidoreductase identification and mutagenesis of two conserved residues that are essential for enzyme activity
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: Helen M Wilks, Michael P. TimkoAbstract:Abstract Protochlorophyllide reductase (NADPH:Protochlorophyllide oxidoreductase; EC 1.6.99.1) catalyzes the light-dependent reduction of Protochlorophyllide to chlorophyllide, a key regulatory step in the chlorophyll biosynthetic pathway. We have developed an expression system in which the Protochlorophyllide reductase from pea (Pisum sativum L.) is used to complement Protochlorophyllide reduction mutants in the photosynthetic bacterium Rhodobacter capsulatus, allowing analysis of wild-type and mutant forms of the enzyme. By protein sequence comparisons, we have identified the plant Protochlorophyllide reductases as belonging to the family of short-chain alcohol dehydrogenases. Based on our protein sequence alignments, we have identified and mutated two conserved residues (Tyr-275 and Lys-279) within the proposed active site of the enzyme and shown that they are critical for activity. A model of the enzyme reaction mechanism for light-dependent Protochlorophyllide reduction is proposed.
Michael Helfrich - One of the best experts on this subject based on the ideXlab platform.
-
pigment free nadph Protochlorophyllide oxidoreductase from avena sativa l
FEBS Journal, 1999Co-Authors: Harald Klement, Michael Helfrich, Ulrike Oster, Siegrid Schoch, Wolfhart RüdigerAbstract:The enzyme NADPH:Protochlorophyllide oxidoreductase (POR) is the key enzyme for light-dependent chlorophyll biosynthesis. It accumulates in dark-grown plants as the ternary enzyme–substrate complex POR-Protochlorophyllide a-NADPH. Here, we describe a simple procedure for purification of pigment-free POR from etioplasts of Avena sativa seedlings. The procedure implies differential solubilization with n-octyl-β-d-glucoside and one chromatographic step with DEAE-cellulose. We show, using pigment and protein analysis, that etioplasts contain a one-to-one complex of POR and Protochlorophyllide a. The preparation of 13 analogues of Protochlorophyllide a is described. The analogues differ in the side chains of the macrocycle and in part contain zinc instead of the central magnesium. Six analogues with different side chains at rings A or B are active substrates, seven analogues with different side chains at rings D or E are not accepted as substrates by POR. The kinetics of the light-dependent reaction reveals three groups of substrate analogues with a fast, medium and slow reaction. To evaluate the kinetic data, the molar extinction coefficients in the reaction buffer had to be determined. At concentrations above 2 mole substrate/mole enzyme, inhibition was found for Protochlorophyllide a and for the analogues.
-
Identification of [8-vinyl]-Protochlorophyllide a in phototrophic prokaryotes and algae: chemical and spectroscopic properties
Biochimica et Biophysica Acta, 1999Co-Authors: Michael Helfrich, Alfred Ross, Garry C. King, Athol G. Turner, Anthony W. D. LarkumAbstract:Abstract [8-vinyl]-Protochlorophyllide a 1 was isolated from a Prochloron sp. associated with the host ascidian, Lissoclinum patella. To obtain sufficient amounts for identification of the purified pigment, suitable extraction procedures and HPLC systems were developed. The structure was finally elucidated by UV-VIS and fluorescence spectroscopy, mass spectrometry and NMR (rotating-frame Overhauser enhancement spectroscopy). [8-vinyl]-Protochlorophyllide a was originally detected only as an intermediate in chlorophyll biosynthesis. Although its presence as a light-harvesting pigment was previously suggested in some prochlorophytes and eukaryotic algae, this is the first unequivocal demonstration of [8-vinyl]-Protochlorophyllide a in an oxygenic phototroph. We also show that [8-vinyl]-Protochlorophyllide a occurs in Prochloron species of four other ascidians as well as in Micromonas pusilla and Prochlorococcus marinus. The possible role of this pigment in photosynthesis is discussed.
-
Photoreduction of zinc protopheophorbide b with NADPH-Protochlorophyllide oxidoreductase from etiolated wheat (Triticum aestivum L.).
European journal of biochemistry, 1995Co-Authors: Siegrid Schoch, Michael Helfrich, Wolfhart Rüdiger, Christer Sundqvist, Bengt Wiktorsson, Margareta RybergAbstract:A preparation of prolamellar bodies from wheat etioplasts was used as a source for NADPH-Protochlorophyllide oxidoreductase (pchlide reductase). The enzyme was solubilized with Triton X-100 after reduction of the endogenous photoconvertible Protochlorophyllide a to chlorophyllide a by saturating illumination. Protochlorophylls a and b, Protochlorophyllide a and zinc protopheophorbide b were added to the soluble enzyme preparation to determine if they were reduced in the dark or in the light. None of the compounds were reduced (with NADPH) in the dark; however, light-dependent reduction was demonstrated with Protochlorophyllide a and zinc protopheophorbide b. The yield was approximately 50% for both substrates. Photoreduction did not occur with the esterified protochlorophylls a and b. Photoreduction of zinc protopheophorbide b, the zinc analogue of Protochlorophyllide b, is the first demonstration of the reduction of a chlorophyll-b-related compound by pchlide reductase.
