Chlorophylls and Bacteriochlorophylls: Biochemistry, Biophysics, Functions and Applications

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Cloning and characterisation of chlorophyll synthase from Avena sativa

The chlorophyll synthase gene from oat (Avena sativa) was cloned and expressed in Escherichia coli. The deduced amino acid sequence consists of 378 amino acids including a presequence, of 46 amino acids. Deletion mutants show that a core protein comprising amino acid residues 88 to 377 is enzymatically active. The sequence of the mature protein shows 85% identity with the chlorophyll synthase of Arabidopsis thaliana and 62% identity with the chlorophyll synthase of Synechocystis PCC 6803. The gene is constitutively expressed as the same transcript level is found in dark-grown and in light-grown seedlings. The enzyme requires magnesium ions for activity; manganese ions can reconstitute only part of the activity. Diacetyl and N-phenylmaleimide (NPM) inhibit the enzyme activity. Site-directed mutagenesis reveals that, out of the 4 Arg residues present in the active core protein, Arg-91 and Arg-161 are essential for the activity. Five cysteine residues are present in the core protein, of which only Cys-109 is essential for the enzyme activity. Since the wild-type and all other Cys-mutants with the exception of the mutant C304A are inhibited by N-phenylmaleimide, we conclude that the inhibitor binds to a non-essential Cys residue to abolish activity. The role of the various Arg and Cys residues is discussed

Inhibition of geranylgeranyl diphosphate synthesis in in vitro systems

The incorporation of [14C]mevalonate and [14C]isopentenyl diphosphate into geranylgeranyl diphosphate was investigated in in vitro systems from Cucurbita pepo (pumpkin) endosperm and from Avena sativa etioplasts. Mevalonate incorporation was effectively inhibited in the pumpkin system by geranylgeranyl diphosphate and geranylgeranyl monophosphate but less effectively by phytyl diphosphate or inorganic diphosphate. Membrane lipids, geranyllinalool, or lecithin enhanced mevalonate incorporation in the Cucurbita system. Incorporation of isopentenyl diphosphate was also enhanced by lecithin and inhibited by geranylgeranyl diphosphate in the Cucurbita system. No lipid enhancement was found in the Avena system; inhibition by GGPP required a much higher GGPP concentration than in the Cucurbita system

Dimerization of the plant photoreceptor phototropin is probably mediated by the LOV1 domain

AbstractPhototropin is a membrane-bound UV-A/blue light photoreceptor of plants responsible for phototropism, chloroplast migration and stomatal opening. Characteristic are two LOV domains, each binding one flavin mononucleotide, in the N-terminal half and having a serine/threonine kinase domain in the C-terminal half of the molecule. We purified the N-terminal half of oat phototropin 1, containing LOV1 and LOV2 domains, as a soluble fusion protein with the calmodulin binding peptide (CBP) by expression in Escherichia coli. Gel chromatography showed that it was dimeric in solution. While the fusion protein CBP-LOV2 was exclusively monomeric in solution, the fusion protein CBP-LOV1 occurred as monomer and dimer. The proportion of dimer increased on prolonged incubation. We conclude that native phototropin is a dimer and that the LOV1 domain is probably responsible for dimerization

In Vitro Synthesis of Chlorophyll A in the Dark Triggers Accumulation of Chlorophyll A Apoproteins in Barley Etioplasts”

An in vitro translation system using lysed etioplasts was developed to test if the accumulation of plastid-encoded chlorophyll a apoproteins is dependent on the de novo synthesis of chlorophyll a. The P700 apoproteins, CP47 and CP43, were not radiolabeled in pulsechase translation assays employing lysed etioplasts in the absence of added chlorophyll precursors. When chlorophyllide a plus phytylpyrophosphate were added to lysed etioplast translation assays in the dark, chlorophyll a was synthesized and radiolabeled P700 apoproteins, CP47 and CP43, and a protein which comigrates with D1 accumulated. Chlorophyllide a or phytylpyrophosphate added separately to the translation assay in darkness did not induce chlorophyll a formation or chlorophyll a apoprotein accumulation. Chlorophyll a formation and chlorophyll a apoprotein accumulation were also induced in the lysed etioplast translation system by the photoreduction of protochlorophyllide to chlorophyllide a in the presence of exogenous phytylpyrophosphate. Accumulation of radiolabeled CP47 was detectable when very low levels of chlorophyll a were synthesized de novo (less than 0.01 nmol/10(7) plastids), and radiolabel increased linearly with increasing de novo chlorophyll a formation. Higher levels of de novo synthesized chlorophyll a were required prior to detection of radiolabel incorporation into the P700 apoproteins and CP43 (greater than 0.01 nmol/10(7) plastids). Radiolabel incorporation into the P700 apoproteins, CP47 and CP43, saturated at a chlorophyll a concentration which corresponds to 50% of the etioplast protochlorophyllide content (0.06 nmol of chlorophyll a/10(7) plastids)

Isolation of the Phytochrome Chromophore. The Cleavage Reaction with Hydrogen Bromide

The cleavage of the bilin chromophore from C-phycocyanin with hydrogen bromide yields 3E-configurated phycocyanobilin (4) as the major and 3 Z-configurated phycocyanobilin (5) as the minor reaction product. The reaction of synthetic 3E-configurated phytochromobilin (2) with hydrogen bromide and methanol leads only to a methanol adduct at the C-18 side chain (7) whereas the same reaction with the 3Z-configurated phytochromobilin (3) leads to 7 and 2. The bilin chromophore was cleaved also from phytochrome after preparation of phytochromobilin peptides. The detection of 2 and 7 suggested that 3Z-and 3E-configurated phytochromobilin were the primary products of cleavage from phytochrome. A reaction scheme is given which can explain the results of the reaction with hydrogen bromide and methanol

Chlorophylls of the c family: absolute configuration and inhibition of NADPH:protochlorophyllide oxidoreductase

AbstractUsing circular dichroism (CD) spectroscopy, the stereochemistry at C-132 of members of the chlorophyll (Chl) c family, namely Chls c1, c2, c3 and [8-vinyl]-protochlorophyllide a (Pchlide a) was determined. By comparison with spectra of known enantiomers, all Chl c members turned out to have the (R) configuration, which is in agreement with considerations drawn from chlorophyll biosynthesis. Except for a double bond in the side chain at C-17, the chemical structure of Chl c1 is identical with Pchlide a, the natural substrate of the light-dependent NADPH:protochlorophyllide oxidoreductase (POR). Thus, lack of binding to the active site due to the wrong configuration at C-132, which had been proposed previously, cannot be an explanation for inactivity of Chl c in this enzymic reaction. Our results show rather that Chl c1 is a competitive inhibitor for this enzyme, tested with Pchlide a and Zn-protopheophorbide a (Zn-Ppheide a) as substrates