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

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Abstract

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

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Abbreviations

AHI:

7-α-Hydroxysteroid dehydrogenase

5-ALA:

5-Aminolevulinic acid

EPR:

Electron paramagnetic resonance

FRET:

Fluorescence resonance energy transfer

LHPP:

Light-harvesting POR-Pchlide complex

Pchlide:

Protochlorophyllide

Chlide:

Chlorophyllide

POR:

NADPH:protochlorophyllide oxidoreductase

PAGE:

Polyacryl-amide gel electrophoresis

SCD:

Short chain dehydrogenase

ZnPP:

Zn protopheophorbide

References

  • Alonso JM et al (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301:653–657

    Article  PubMed  Google Scholar 

  • Apel K (1981) The protochlorophyllide holochrome of barley (Hordeum vulgare L.): phytochrome-induced decrease of translatable mRNA coding for the NADPH:protochlorophyllide oxidoreductase. Eur J Biochem 120:89–93

    Article  PubMed  CAS  Google Scholar 

  • Apel K, Santel H-J, Redlinger TE, Falk H (1980) The protochlorophyllide holochrome of barley. Isolation and characterization of the NADPH:protochlorophyllide oxidoreductase. Eur J Biochem 111:251–258

    Article  PubMed  CAS  Google Scholar 

  • Armstrong GA, Apel K, Rüdiger W (2000) Does a light-harvesting protochlorophyllide a/b binding protein complex exist? Trends Plant Sci 5:40–44

    Article  PubMed  CAS  Google Scholar 

  • Baker ME (1994) Protochlorophyllide oxidoreductase is homologous to human carbonyl reductase and pig 20β-hydroxysteroid dehydrogenase. Biochem J 300:605–607

    PubMed  CAS  Google Scholar 

  • Bauer J, Hiltbrunner A, Kessler F (2001) Molecular biology of chloroplast biogenesis: gene expression, protein import and intraorganellar sorting. Cell Mol Life Sci 58:420–433

    Article  PubMed  CAS  Google Scholar 

  • Birve SJ, Selstam E, Johansson BA (1996) Secondary structure of NADPH:protochlorophyllide oxidoreductase examined by circular dichroism and prediction methods. Biochem J 317:549–555

    PubMed  CAS  Google Scholar 

  • Boardman NK (1962) Studies on a protochlorophyll–protein complex. I. Purification and molecular weight determination. Biochem Biophys Acta 62:63–79

    Article  PubMed  CAS  Google Scholar 

  • Cline K, Werner-Washburne M, Andrews J, Keegstra K (1984) Thermolysin is a suitable protease for probing the surface of intact pea chloroplasts. Plant Physiol 75:675–678

    PubMed  CAS  Google Scholar 

  • Crowe LM, Crowe JH (1984) Preservation of membranes in anhydrobiotic organisms: the role of trehalose. Science 223:701–703

    Article  PubMed  CAS  Google Scholar 

  • Crowe LM, Mouradian R, Crowe JH, Jackson SA, Womersley C (1984) Effects of carbohydrates on membrane stability at low water potentials. Biochim Biophys Acta 769:141–150

    Article  PubMed  CAS  Google Scholar 

  • Dahlin C, Aronsson H, Wilks HM, Lebedev N, Sundqvist C, Timko MP (1999) 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. Plant Mol Biol 39:309–323

    Article  PubMed  CAS  Google Scholar 

  • Dehesh K, Ryberg M (1985) The NADPH-protochlorophyllide oxidoreductase is the major protein constituent of prolamellar bodies in wheat (Triticum aestivum L.). Planta 164:396–399

    Article  CAS  Google Scholar 

  • Franck F, Sperling U, Frick H, Pochert B, van Cleve B, Apel K, Armstrong GA (2000) Regulation of etioplast pigment-protein complexes, inner membrane architecture, and protochloro-phyllide a chemical heterogeneity by light-dependent NADPH:protochlorophyllide oxidoreductases A and B. Plant Physiol 124:1678–1696

    Article  PubMed  CAS  Google Scholar 

  • Frick G, Su Q, Apel K, Armstrong GA (2003) An Arabidopsis porB porC double mutant lacking light-dependent NADPH:protochlorophyllide oxidoreductases B and C is highly chlorophyll-deficient and developmentally arrested. Plant J 25:141–153

    Article  CAS  Google Scholar 

  • Griffiths TW (1975) Characterization of the terminal stage of chlorophyll(ide) synthesis in etioplast membrane preparations. Biochem J 152:621–635

    Google Scholar 

  • Griffiths TW (1978) Reconstitution of chlorophyllide formation by isolated etioplast membranes. Biochem J 174:681–692

    PubMed  CAS  Google Scholar 

  • Holtorf H, Reinbothe S, Reinbothe C, Bereza B, Apel K (1995) Two routes of chlorophyllide synthesis that are differentially regulated by light in barley. Proc Natl Acad Sci USA 92:3254–3258

    Article  PubMed  CAS  Google Scholar 

  • Ignatov NV, Litvin FF (1981) Energy migration in a pigmented protochlorophyllide complex. Biofizika 26:664–668

    PubMed  CAS  Google Scholar 

  • Kahn A (1968) Developmental physiology of bean leaf plastids. Tube transformation and protochlorphyll(ide) photoconversion by a flash irradiation. Plant Physiol 43:1781–1785

    PubMed  CAS  Google Scholar 

  • Kahn AA, Boardman NK, Thorne SW (1970) Energy transfer between protochlorophyllide molecules: evidence for multiple chromophores in the photoactive protochlorophyllide-protein complex in vivo and in vitro. J Mol Biol 48:85–101

    Article  PubMed  CAS  Google Scholar 

  • Krieg PA, Melton DA (1984) Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs. Nucleic Acids Res 12:7057–7070

    Article  PubMed  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  PubMed  CAS  Google Scholar 

  • Lebedev N, Timko MP (1999) Protochlorophyllide oxidoreductase B-catalyzed protochlorophyllide photoreduction in vitro: insight into the mechanism of chlorophyll formation in light-adapted plants. Proc Natl Acad Sci USA 96:9954–9959

    Article  PubMed  CAS  Google Scholar 

  • Lebedev N, Karginova O, Mcivor W, Timko MP (2001) Tyr275 and Lys279 stabilize NADPH within the catalytic site of NADPH:protochlorophyllide oxidoreductase and are involved in the formation of the enzyme photoactive state. Biochemistry 40:12562–12574

    Article  PubMed  CAS  Google Scholar 

  • Mathis P, Sauer K (1972) Circular dichroism studies on the structure and the photochemistry of protochlorophyllide and chlorophyllide holochrome. Biochim Biophys Acta 267:498–511

    Article  PubMed  CAS  Google Scholar 

  • Oliver PR, Griffiths TW (1981) Covalent labelling of the NADPH:protochlorophyllide oxidoreductase from etioplast membranes with [3H]N-phenylmaleimide. Biochem J 195:93–101

    PubMed  CAS  Google Scholar 

  • Oosawa N, Masuda T, Awai K, Fusada N, Shimada H, Ohta H, Takamiya KI (2000) Identification and light-induced expression of a novel gene of NADPH-protochlorophyllide oxidoreductase isoform in Arabidopsis thaliana. FEBS Lett 474:133–136

    Article  PubMed  CAS  Google Scholar 

  • Pattanayak GK, Tripathy BC (2002) Catalytic function of a novel protein protochlorophyllide oxidoreductase C of Arabidopsis thaliana. Biochem Biophys Res Commun 291:921–924

    Article  PubMed  CAS  Google Scholar 

  • Reinbothe S, Krauspe R, Parthier B (1990) In vitro-import of chloroplast proteins in a homologous Euglena system with particular reference to plastid leucyl-tRNA synthetase. Planta 181:176–183

    Article  CAS  Google Scholar 

  • Reinbothe S, Reinbothe C, Holtorf H, Apel K (1995a) Two NADPH:protochlorophyllide oxidoreductases in barley: evidence for the selective disappearance of PORA during the light-induced greening of etiolated seedlings. Plant Cell 7:1933–1940

    Article  CAS  Google Scholar 

  • Reinbothe S, Runge S, Reinbothe C, van Cleve B, Apel K (1995b) Substrate-dependent transport of the NADPH:protochloro-phyllide oxidoreductase into isolated plastids. Plant Cell 7:161–172

    Article  CAS  Google Scholar 

  • Reinbothe C, Apel K, Reinbothe S (1995c) A light-induced protease from barley plastids degrades NADPH:protochlorophyllide oxidoreductase complexed with chlorophyllide. Mol Cell Biol 15:6206–6212

    CAS  Google Scholar 

  • Reinbothe S, Reinbothe C, Lebedev N, Apel K (1996) PORA and PORB, two light-dependent protochlorophyllide-reducing enzymes of angiosperm chlorophyll biosynthesis. Plant Cell 8:763–769

    Article  PubMed  CAS  Google Scholar 

  • Reinbothe C, Lebedev N, Apel K, Reinbothe S (1997) Regulation of chloroplast protein import through a protochlorophyllide-responsive transit peptide. Proc Natl Acad Sci USA 94:8890–8894

    Article  PubMed  CAS  Google Scholar 

  • Reinbothe C, Lebedev N, Reinbothe S (1999) A protochloro-phyllide light-harvesting complex involved in de-etiolation of higher plants. Nature 397:80–84

    Article  CAS  Google Scholar 

  • Reinbothe C, Mache R, Reinbothe S (2000) A second, substrate-dependent site of protein import into chloroplasts. Proc Natl Acad Sci USA 97:9795–9800

    Article  PubMed  CAS  Google Scholar 

  • Reinbothe C, Buhr F, Pollmann S, Reinbothe S (2003a) In vitro-reconstitution of LHPP with protochlorophyllides a and b. J Biol Chem 278:807–815

    Article  CAS  Google Scholar 

  • Reinbothe S, Pollmann S, Reinbothe C (2003b) In-situ-conversion of protochlorophyllide b to protochlorophyllide a in barley. Evidence for a novel role of 7-formyl reductase in the prolamellar body of etioplasts. J Biol Chem 278:800–806

    Article  CAS  Google Scholar 

  • Sanger F, Nickler S, Coulsen A R (1977) DNA sequencing with chain-termination inhibitors. Proc Natl Acad Sci USA 74:5463–5467

    Article  PubMed  CAS  Google Scholar 

  • Smith JHC, Benitez A (1954) The effect of temperature on the conversion of protochlorophyll to chlorophyll a in etiolated barley leaves. Plant Physiol 29:135–143

    Article  PubMed  CAS  Google Scholar 

  • Su Q, Frick G, Armstrong G, Apel K (2001) PORC of Arabidopsis thaliana: a third light- and NADPH-dependent protochlorophyllide oxidoreductase that is differentially regulated by light. Plant Mol Biol 47:805–813

    Article  PubMed  CAS  Google Scholar 

  • Townley HE, Sessions RB, Clarke AR, Dafforn TR, Griffith TW (2001) Protochlorophyllide oxidoreductase: a homology model examined by site-directed mutagenesis. Proteins Struct Funct Genet 44:329–335

    Article  PubMed  CAS  Google Scholar 

  • Virgin HI, Kahn A, von Wettstein D (1963) The physiology of chlorophyll formation in relation to structural changes in chloroplasts. Photochem Photobiol 2:83–91

    Article  CAS  Google Scholar 

  • Wilks H, Timko MP (1995) A light-dependent complementation system for analysis of NADPH:protochlorophyllide oxidoreductase: identification and mutagenesis of two conserved residues that are essential for enzyme activity. Proc Natl Acad Sci USA 92:724–728

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Lehrstuhl für Pflanzenphysiologie, Universität Bayreuth, Universitätsstraße 30, 95447, Bayreuth, Germany

    Christiane Reinbothe, Frank Buhr & Sandra Bartsch

  2. Université Joseph Fourier et Centre National de la Recherche Scientifique, Laboratoire de Génétique moléculaire des Plantes, UMR 5575, CERMO, BP53, 38041, Grenoble cedex 9, France

    Claire Desvignes, Françoise Quigley, Hélène Pesey & Steffen Reinbothe

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  1. Christiane Reinbothe
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  2. Frank Buhr
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  3. Sandra Bartsch
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  4. Claire Desvignes
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  6. Hélène Pesey
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  7. Steffen Reinbothe
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Correspondence to Christiane Reinbothe.

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Communicated by R. Herrmann

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Reinbothe, C., Buhr, F., Bartsch, S. et al. In vitro-mutagenesis of NADPH:protochlorophyllide oxidoreductase B: two distinctive protochlorophyllide binding sites participate in enzyme catalysis and assembly. Mol Genet Genomics 275, 540–552 (2006). https://doi.org/10.1007/s00438-006-0109-9

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  • DOI: https://doi.org/10.1007/s00438-006-0109-9

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