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Query: UNIPROT:P06889 (Mol)
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NADPH: protochlorophyllide oxidoreductase (pchlide reductase, EC 1.6.99.1) catalyzes the light-dependent reduction of protochlorophyllide in higher plants. Cloned cDNAs encoding two distinct pchlide reductases were isolated from a lambda gt11 library constructed from poly(A)+ RNA prepared from the cotyledons of dark-grown white pine (Pinus strobus) seedlings and a nuclear gene (lpcr) analogous to one of these cDNAs has been characterized from loblolly pine (P. taeda). The pine gene encodes an approximately 43 kDa precursor polypeptide consisting of a 334-amino acid mature protein and a 66-amino acid transit peptide. The deduced primary structures for the pine proteins are highly homologous to those reported from monocots and dicots. The coding portion of the pine lpcr gene is interrupted by four introns. The placement of these introns within the pine lpcr gene is identical to that observed in pea (Pisum sativum), suggesting conservation in gene organization between dicot and gymnosperm species. Western blot analysis using polyclonal antiserum against oat pchlide reductase detected in extracts of dark-grown pine cotyledons a single immunoreactive protein, which declined in abundance during a 48 h period of illumination with white light. Cotyledons of dark-grown seedlings were also found to accumulate high levels of pchlide reductase mRNA; however, little or no change in the steady-state levels of mRNA encoding pchlide reductase was observed in these tissues following illumination. Stem tissue of dark-grown seedlings did not contain significant levels of pchlide reductase mRNA, whereas stems of light-grown plants of the same age accumulated substantial amounts of the message. These results suggest that light and the developmental age of the tissue affect regulation of lpcr expression in pine.
Mol Gen Genet 1992 Dec
PMID:NADPH: protochlorophyllide oxidoreductases in white pine (Pinus strobus) and loblolly pine (P. taeda). Evidence for light and developmental regulation of expression and conservation in gene organization and protein structure between angiosperms and gymnosperms. 149 55

Complementary DNA clones and a corresponding nuclear gene (lpcr) encoding the NADPH-dependent protochlorophyllide oxidoreductase (pchlide reductase, EC 1.6.99.1) have been characterized from pea (Pisum sativum L.). The pea lpcr gene encodes a 43,118 Da precursor polypeptide comprised of a transit peptide of 64 amino acids and a mature protein of 336 amino acids. The coding portion of the gene is interrupted by four introns, two of which are located within the transit peptide coding portion of the gene. The deduced primary structure for the pea protein is similar to those reported for Arabidopsis and two monocot species. Northern blot analysis revealed little to no decrease in steady-state levels of mRNA encoding the enzyme in etiolated leaves illuminated with continuous white light for up to 48 h. In contrast, western blot analysis showed that the major immunoreactive species present in whole leaf extracts decreased to nearly undetectable levels during this same 48 h period. These results suggest that pchlide reductase activity in pea is primarily regulated post-transcriptionally, most likely at the level of translation initiation/elongation or protein turnover.
Plant Mol Biol 1992 Mar
PMID:Molecular cloning, nuclear gene structure, and developmental expression of NADPH: protochlorophyllide oxidoreductase in pea (Pisum sativum L.). 158 73

A cDNA encoding the NADPH-protochlorophyllide oxidoreductase (Pchlide reductase) of Arabidopsis thaliana has been isolated and sequenced. The cDNA contains the complete reading frame for the precursor of the Pchlide reductase. The deduced amino acid sequence of the Arabidopsis enzyme closely resembles the corresponding sequences of barley and oat. The cDNA has been used as a template for the synthesis of the enzyme protein in Escherichia coli. An antiserum was raised against this enzyme protein and both the antiserum and the cDNA were used as experimental tools to study the effects of light on the Pchlide reductase in A. thaliana. When etiolated seedlings of Arabidopsis were exposed to light the enzyme activity and the concentration of the enzyme protein rapidly declined. Similar light effects have been described previously for other angiosperms. In contrast to most of these species, however, in Arabidopsis only minor changes in Pchlide reductase mRNA content could be observed when etiolated seedlings were exposed to light.
Plant Mol Biol 1991 Apr
PMID:Effect of light on the NADPH-protochlorophyllide oxidoreductase of Arabidopsis thaliana. 171 19

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.
Mol Gen Genet 1989 Jun
PMID:Nucleotide sequence of a cDNA coding for the NADPH-protochlorophyllide oxidoreductase (PCR) of barley (Hordeum vulgare L.) and its expression in Escherichia coli. 267 59

The NADPH:protochlorophyllide oxidoreductase precursor protein (pPorA) of barley (Hordeum vulgare L. cv. Carina), synthesized from a full-length cDNA clone by coupling in vitro transcription and translation, is a catalytically active protein. It converts protochlorophyllide to chlorophyllide in a light- and NADPH-dependent manner. At least the pigment product of catalysis remains tightly bound to the precursor protein. The chlorophyllide-pPorA complex differs markedly from the protochlorophyllide-pPorA complex with respect to sensitivity to attack by a light-induced, nucleus-encoded, and energy-dependent protease activity of barley plastids. The pPorA-chlorophyllide complex is rapidly degraded, in contrast to pPorA-protochlorophyllide complexes containing or lacking NADPH, which are both resistant to protease treatment. Unexpectedly, pPorA devoid of its substrates or products was less sensitive to proteolysis than the pPorA-chlorophyllide complex, suggesting that both substrate binding and product formation during catalysis had caused differential changes in protein conformation.
Mol Cell Biol 1995 Nov
PMID:A light-induced protease from barley plastids degrades NADPH:protochlorophyllide oxidoreductase complexed with chlorophyllide. 756 73

The NADPH-protochlorophyllide oxidoreductase (pchlide reductase, EC 1.6.99.1) is the major protein in the prolamellar bodies (PLBs) of etioplasts, where it catalyzes the light-dependent reduction of protochlorophyllide to chlorophyllide during chlorophyll synthesis in higher plants. The suborganellar location in chloroplasts of light-grown plants is less clear. In vitro assays were performed to characterize the assembly process of the pchlide reductase protein in pea chloroplasts. Import reactions employing radiolabelled precursor protein of the pchlide reductase showed that the protein was efficiently imported into fully matured green chloroplasts of pea. Fractionation assays following an import reaction revealed that imported protein was targeted to the thylakoid membranes. No radiolabelled protein could be detected in the stromal or envelope compartments upon import. Assembly reactions performed in chloroplast lysates showed that maximum amount of radiolabelled protein was associated to the thylakoid membranes in a thermolysin-resistant conformation when the assays were performed in the presence of hydrolyzable ATP and NADPH, but not in the presence of NADH. Furthermore, membrane assembly was optimal at pH 7.5 and at 25 degrees C. However, further treatment of the thylakoids with NaOH after an assembly reaction removed most of the membrane-associated protein. Assembly assays performed with the mature form of the pchlide reductase, lacking the transit peptide, showed that the pre-sequence was not required for membrane assembly. These results indicate that the pchlide reductase is a peripheral protein located on the stromal side of the membrane, and that both the precursor and the mature form of the protein can act as substrates for membrane assembly.
Plant Mol Biol 1995 Oct
PMID:The in vitro assembly of the NADPH-protochlorophyllide oxidoreductase in pea chloroplasts. 757 82

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. Similarly, 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.
Plant Mol Biol 1996 Jan
PMID:The pc-1 phenotype of Chlamydomonas reinhardtii results from a deletion mutation in the nuclear gene for NADPH:protochlorophyllide oxidoreductase. 861 32

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.
Plant Mol Biol 1996 Jun
PMID: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. 879 Feb 86

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.
Plant Mol Biol 1999 Jan
PMID: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. 1008 Jun 97

The expression patterns of the two distinct subfamilies of genes (designated porA and porB) encoding the light-dependent NADPH:protochlorophyllide oxidoreductases (PORs) in loblolly pine (Pinus taeda L.) were examined. Transcripts arising from both gene subfamilies were shown to be present at high levels in the cotyledons of dark-grown pine seedlings and to a lesser extent in their stems. Exposure of dark-grown seedlings to light resulted in increased levels of both porA and porB transcripts, as well as increased levels of mRNAs encoding other photosynthesis-related gene products, suggesting that they are under a common mode of regulation. Relative levels of the porA and porB transcripts were similar in seedling cotyledons and primary needles of two-month-old pine trees, whereas only porB transcripts were present at a significant level in mature secondary needles of two-year-old trees. Immunoblot analysis showed that the 37 kDa PORA protein was most abundant in dark-grown tissues, decreased dramatically upon exposure to light, but could still be detected at low levels in light-grown seedlings. In comparison, levels of the 38 kDa PORB protein were not significantly changed upon transfer of dark-grown tissues to light. While both PORA and PORB were detected in cotyledons and primary needles, only PORB could be detected in mature needles. Transcripts derived from the three plastid genes, chlL, chlN, and chlB, encoding subunits of the light-independent protochlorophyllide reductase were detected in the cotyledons and stems of dark-grown seedlings, and in mature needles. The highest levels of chlL, chlN, and chlB transcripts were detected within the top one-third of the stem and decreased gradually towards the stem/root transition zone. Correspondingly, the highest levels of light-independent chlorophyll formation took place near the top of the hypocotyl. A similar pattern of expression was observed for other photosynthesis-related gene products, including porA and porB. Our results suggest that many aspects of the light-dependent, tissue-specific and developmental regulation of POR expression first described in angiosperms were already established in the less evolutionarily advanced gymnosperms. However, unlike angiosperms, light is not the dominant regulatory factor controlling porA expression in these species.
Plant Mol Biol 1999 Feb
PMID:Differential expression of genes encoding the light-dependent and light-independent enzymes for protochlorophyllide reduction during development in loblolly pine. 1009 84


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