Publications

• Identification of a vinyl reductase gene for chlorophyll synthesis in Arabidopsis thaliana and implications for the evolution of Prochlorococcus species.

  Nagata N., Tanaka R., Satoh S., Tanaka A.

  Chlorophyll metabolism has been extensively studied with various organisms, and almost all of the chlorophyll biosynthetic genes have been identified in higher plants. However, only the gene for 3,8-divinyl protochlorophyllide a 8-vinyl reductase (DVR), which is indispensable for monovinyl chlorop ... >> More

  Chlorophyll metabolism has been extensively studied with various organisms, and almost all of the chlorophyll biosynthetic genes have been identified in higher plants. However, only the gene for 3,8-divinyl protochlorophyllide a 8-vinyl reductase (DVR), which is indispensable for monovinyl chlorophyll synthesis, has not been identified yet. In this study, we isolated an Arabidopsis thaliana mutant that accumulated divinyl chlorophyll instead of monovinyl chlorophyll by ethyl methanesulfonate mutagenesis. Map-based cloning of this mutant resulted in the identification of a gene (AT5G18660) that shows sequence similarity with isoflavone reductase genes. The mutant phenotype was complemented by the transformation with the wild-type gene. A recombinant protein encoded by AT5G18660 was expressed in Escherichia coli and found to catalyze the conversion of divinyl chlorophyllide to monovinyl chlorophyllide, thereby demonstrating that the gene encodes a functional DVR. DVR is encoded by a single copy gene in the A. thaliana genome. With the identification of DVR, finally all genes required for chlorophyll biosynthesis have been identified in higher plants. Analysis of the complete genome of A. thaliana showed that it has 15 enzymes encoded by 27 genes for chlorophyll biosynthesis from glutamyl-tRNA(glu) to chlorophyll b. Furthermore, identification of the DVR gene helped understanding the evolution of Prochlorococcus marinus, a marine cyanobacterium that is dominant in the open ocean and is uncommon in using divinyl chlorophylls. A DVR homolog was not found in the genome of P. marinus but found in the Synechococcus sp WH8102 genome, which is consistent with the distribution of divinyl chlorophyll in marine cyanobacteria of the genera Prochlorococcus and Synechococcus. << Less

  Plant Cell 17:233-240(2005) [PubMed] [EuropePMC]

• Divinyl chlorophyll(ide) a can be converted to monovinyl chlorophyll(ide) a by a divinyl reductase in rice.

  Wang P., Gao J., Wan C., Zhang F., Xu Z., Huang X., Sun X., Deng X.

  3,8-Divinyl (proto)chlorophyll(ide) a 8-vinyl reductase (DVR) catalyzes the reduction of 8-vinyl group on the tetrapyrrole to an ethyl group, which is indispensable for monovinyl chlorophyll (Chl) synthesis. So far, three 8-vinyl reductase genes (DVR, bciA, and slr1923) have been characterized fro ... >> More

  3,8-Divinyl (proto)chlorophyll(ide) a 8-vinyl reductase (DVR) catalyzes the reduction of 8-vinyl group on the tetrapyrrole to an ethyl group, which is indispensable for monovinyl chlorophyll (Chl) synthesis. So far, three 8-vinyl reductase genes (DVR, bciA, and slr1923) have been characterized from Arabidopsis (Arabidopsis thaliana), Chlorobium tepidum, and Synechocystis sp. PCC6803. However, no 8-vinyl reductase gene has yet been identified in monocotyledonous plants. In this study, we isolated a spontaneous mutant, 824ys, in rice (Oryza sativa). The mutant exhibited a yellow-green leaf phenotype, reduced Chl level, arrested chloroplast development, and retarded growth rate. The phenotype of the 824ys mutant was caused by a recessive mutation in a nuclear gene on the short arm of rice chromosome 3. Map-based cloning of this mutant resulted in the identification of a gene (Os03g22780) showing sequence similarity with the Arabidopsis DVR gene (AT5G18660). In the 824ys mutant, nine nucleotides were deleted at residues 952 to 960 in the open reading frame, resulting in a deletion of three amino acid residues in the encoded product. High-performance liquid chromatography analysis of Chls indicated the mutant accumulates only divinyl Chl a and b. A recombinant protein encoded by Os03g22780 was expressed in Escherichia coli and found to catalyze the conversion of divinyl chlorophyll(ide) a to monovinyl chlorophyll(ide) a. Therefore, it has been confirmed that Os03g22780, renamed as OsDVR, encodes a functional DVR in rice. Based upon these results, we succeeded to identify an 8-vinyl reductase gene in monocotyledonous plants and, more importantly, confirmed the DVR activity to convert divinyl Chl a to monovinyl Chl a. << Less

  Plant Physiol. 153:994-1003(2010) [PubMed] [EuropePMC]

• Chloroplast biogenesis 84: solubilization and partial purification of membrane-bound [4-vinyl]chlorophyllide a reductase from etiolated barley leaves.

  Kolossov V.L., Rebeiz C.A.

  [4-Vinyl] chlorophyllide a reductase (4VCR) is a key enzyme of the chlorophyll (Chl) biosynthetic pathway. It catalyzes the conversion of divinyl chlorophyllide (Chlide) a to monovinyl Chlide a by reduction of the vinyl group at position 4 of the macrocycle to ethyl. 4VCR is a membrane-bound enzym ... >> More

  [4-Vinyl] chlorophyllide a reductase (4VCR) is a key enzyme of the chlorophyll (Chl) biosynthetic pathway. It catalyzes the conversion of divinyl chlorophyllide (Chlide) a to monovinyl Chlide a by reduction of the vinyl group at position 4 of the macrocycle to ethyl. 4VCR is a membrane-bound enzyme, embedded in etioplast and etiochloroplast membranes. A study of the regulation and properties of this enzyme is mandatory for a comprehensive understanding of the biosynthetic heterogeneity of Chl biosynthesis. Solubilization and partial purification of 4VCR are described for the first time. The enzyme was solubilized with 5 mM Chaps and was partially purified by chromatography on DEAE-Sephacel and Cibacron Blue 3GA-1000 agarose. An overall 20-fold purification was achieved. The partially purified enzyme was stable for several months at -80 degrees C. << Less

  Anal Biochem 295:214-219(2001) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Chloroplast Biogenesis 60 : Conversion of Divinyl Protochlorophyllide to Monovinyl Protochlorophyllide in Green(ing) Barley, a Dark Monovinyl/Light Divinyl Plant Species.

  Tripathy B.C., Rebeiz C.A.

  In higher plants, most of the chlorophyll a is formed via the divinyl and monovinyl chlorophyll monocarboxylic biosynthetic routes. These two routes are strongly interconnected prior to protochlorophyllide formation in barley (Hordeum vulgare L. cv Morex), a dark monovinyl-light divinyl plant spec ... >> More

  In higher plants, most of the chlorophyll a is formed via the divinyl and monovinyl chlorophyll monocarboxylic biosynthetic routes. These two routes are strongly interconnected prior to protochlorophyllide formation in barley (Hordeum vulgare L. cv Morex), a dark monovinyl-light divinyl plant species, but not in cucumber (Cucumis sativus L. cv Beit Alpha MR), a dark divinyl-light divinyl plant species (BC Tripathy, CA Rebeiz, 1986 J Biol Chem 261: 13556-13564). It is shown that in dark monovinyl-light divinyl plant species such as barley, the divinyl and monovinyl monocarboxylic routes become interconnected at the level of protochlorophyllide during transition from the divinyl to the monovinyl protochlorophyllide biosynthetic mode. In cucumber, a dark divinyl-light divinyl plant species, in which the monovinyl monocarboxylic biosynthetic route becomes preponderant only after an abnormally long sojourn in darkness, the conversion of divinyl to monovinyl protochlorophyllide does not take place on the barley time-scale of incubation. << Less

  Plant Physiol 87:89-94(1988) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Chloroplast biogenesis 72: a [4-vinyl]chlorophyllide a reductase assay using divinyl chlorophyllide a as an exogenous substrate.

  Parham R., Rebeiz C.A.

  [4-Vinyl]Chlorophyllide alpha reductase (4VCR) catalyzes the conversion of 2,4-divinyl chlorophyllide alpha (DV Chlide alpha) to 2-vinyl,4-ethyl chlorophyllide alpha (MV Chlide alpha) via an NAPDH-dependent reaction. MV Childe alpha is the immediate precursor of monovinyl chlorophyll alpha in plan ... >> More

  [4-Vinyl]Chlorophyllide alpha reductase (4VCR) catalyzes the conversion of 2,4-divinyl chlorophyllide alpha (DV Chlide alpha) to 2-vinyl,4-ethyl chlorophyllide alpha (MV Chlide alpha) via an NAPDH-dependent reaction. MV Childe alpha is the immediate precursor of monovinyl chlorophyll alpha in plants. In etiolated cucumber (Cucumis sativus L.) cotyledons, 4VCR is a plastidic membrane-bound enzyme. Further research on this enzyme required the development of an assay that utilizes DV Childe alpha as an exogenous substrate. Such an assay is now described. It involves conversion of exogenous DV Chlide alpha to MV Chlide alpha at high rates by etioplast membranes of cucumber, corn (Zea mays L.), and barley (Hordum vulgare L.). 4VCR exhibits high activity between 30 and 40C and in the pH range of 6.3 to 7.0. Activity is quasilinear for the first 60 s of incubation. << Less

  Anal Biochem 231:164-169(1995) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Characterization of a plant-like protochlorophyllide a divinyl reductase in green sulfur bacteria.

  Chew A.G., Bryant D.A.

  The green sulfur bacterium Chlorobium tepidum synthesizes three types of (bacterio)chlorophyll ((B)Chl): BChl a(P), Chl a(PD), and BChl c(F). During the synthesis of all three molecules, a C-8 vinyl substituent is reduced to an ethyl group, and in the case of BChl c(F), the C-8(2) carbon of this e ... >> More

  The green sulfur bacterium Chlorobium tepidum synthesizes three types of (bacterio)chlorophyll ((B)Chl): BChl a(P), Chl a(PD), and BChl c(F). During the synthesis of all three molecules, a C-8 vinyl substituent is reduced to an ethyl group, and in the case of BChl c(F), the C-8(2) carbon of this ethyl group is subsequently methylated once or twice by the radical S-adenosylmethionine enzyme BchQ. The C. tepidum genome contains homologs of two genes, bchJ (CT2014) and CT1063, that are highly homologous to genes, bchJ and AT5G18660, and that have been reported to encode C-8 vinyl reductases in Rhodobacter capsulatus and Arabidopsis thaliana, respectively. To determine which gene product actually encodes a C-8 vinyl reductase activity, the bchJ and CT1063 genes were insertionally inactivated in C. tepidum. All three Chls synthesized by the CT1063 mutant of C. tepidum have a C-8 vinyl group. Using NADPH but not NADH as reductant, recombinant BciA reduces the C-8 vinyl group of 3,8-divinyl-protochlorophyllide in vitro. These data demonstrate that CT1063, renamed bciA, encodes a C-8 divinyl reductase in C. tepidum. The bchJ mutant produces detectable amounts of Chl a(PD), BChl a(P), and BChl c(F), all of which have reduced C-8 substituents, but the mutant cells secrete large amounts of 3,8-divinyl-protochlorophyllide a into the growth medium and have a greatly reduced BChl c(F) content. The results suggest that BchJ may play an important role in substrate channeling and/or regulation of Chl biosynthesis but show that it is not a vinyl reductase. Because only some Chl-synthesizing organisms possess homologs of bciA, at least two types of C-8 vinyl reductases must occur. << Less

  J Biol Chem 282:2967-2975(2007) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Chloroplast biogenesis: [4-vinyl] chlorophyllide a reductase is a divinyl chlorophyllide a-specific, NADPH-dependent enzyme.

  Parham R., Rebeiz C.A.

  Some properties of [4-vinyl] chlorophyllide a reductase are described. This enzyme converts divinyl chlorophyllide a to monovinyl chlorophyllide a. The latter is the immediate precursor of monovinyl chlorophyll a, the main chlorophyll in green plants. [4-Vinyl] chlorophyllide a reductase plays an ... >> More

  Some properties of [4-vinyl] chlorophyllide a reductase are described. This enzyme converts divinyl chlorophyllide a to monovinyl chlorophyllide a. The latter is the immediate precursor of monovinyl chlorophyll a, the main chlorophyll in green plants. [4-Vinyl] chlorophyllide a reductase plays an important role in daylight during the conversion of divinyl protochlorophyllide a to monovinyl chlorophyll a. [4-Vinyl] chlorophyllide a reductase was detected in isolated plastid membranes. Its activity is strictly dependent on the availability of NADPH. Other reductants such as NADH and GSH were ineffective. The enzyme appears to be specific for divinyl chlorophyllide a, and it does not reduce divinyl protochlorophyllide a to monovinyl protochlorophyllide a. The conversion of divinyl protochlorophyllide a to monovinyl protochlorophyllide a has been demonstrated in barley and cucumber etiochloroplasts and appears to be catalyzed by a [4-vinyl] protochlorophyllide a reductase [Tripathy, B.C., & Rebeiz, C.A. (1988) Plant Physiol. 87, 89-94]. On the basis of reductant requirements and substrate specificity, it is possible that two different 4-vinyl reductases may be involved in the reduction of divinyl protochlorophyllide a and divinyl chlorophyllide a to their respective 4-ethyl analogues. << Less

  Biochemistry 31:8460-8464(1992) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• The major route for chlorophyll synthesis includes [3,8-divinyl]-chlorophyllide a reduction in Arabidopsis thaliana.

  Nagata N., Tanaka R., Tanaka A.

  In most reviews on Chl biosynthesis, Chl is described as being synthesized via the route involving the reduction of [3,8-divinyl]-protochlorophyllide a. However, the possibility remains that the conversion of the divinyl form of the Chl intermediate to its monovinyl form takes place at other enzym ... >> More

  In most reviews on Chl biosynthesis, Chl is described as being synthesized via the route involving the reduction of [3,8-divinyl]-protochlorophyllide a. However, the possibility remains that the conversion of the divinyl form of the Chl intermediate to its monovinyl form takes place at other enzymatic steps. To determine the actual route of Chl biosynthesis, we examined the substrate specificity of the formerly named [3,8-divinyl]-protochlorophyllide a 8-vinyl reductase (DVR) in vitro. In addition, we investigated the accumulation of various Chl intermediates in etiolated seedlings in vivo. Collectively, these studies indicate that [3,8-divinyl]-chlorophyllide a is the major substrate of DVR. << Less

  Plant Cell Physiol. 48:1803-1808(2007) [PubMed] [EuropePMC]

• One divinyl reductase reduces the 8-vinyl groups in various intermediates of chlorophyll biosynthesis in a given higher plant species, but the isozyme differs between species.

  Wang P., Wan C., Xu Z., Wang P., Wang W., Sun C., Ma X., Xiao Y., Zhu J., Gao X., Deng X.

  Divinyl reductase (DVR) converts 8-vinyl groups on various chlorophyll intermediates to ethyl groups, which is indispensable for chlorophyll biosynthesis. To date, five DVR activities have been detected, but adequate evidence of enzymatic assays using purified or recombinant DVR proteins has not b ... >> More

  Divinyl reductase (DVR) converts 8-vinyl groups on various chlorophyll intermediates to ethyl groups, which is indispensable for chlorophyll biosynthesis. To date, five DVR activities have been detected, but adequate evidence of enzymatic assays using purified or recombinant DVR proteins has not been demonstrated, and it is unclear whether one or multiple enzymes catalyze these activities. In this study, we systematically carried out enzymatic assays using four recombinant DVR proteins and five divinyl substrates and then investigated the in vivo accumulation of various chlorophyll intermediates in rice (Oryza sativa), maize (Zea mays), and cucumber (Cucumis sativus). The results demonstrated that both rice and maize DVR proteins can convert all of the five divinyl substrates to corresponding monovinyl compounds, while both cucumber and Arabidopsis (Arabidopsis thaliana) DVR proteins can convert three of them. Meanwhile, the OsDVR (Os03g22780)-inactivated 824ys mutant of rice exclusively accumulated divinyl chlorophylls in its various organs during different developmental stages. Collectively, we conclude that a single DVR with broad substrate specificity is responsible for reducing the 8-vinyl groups of various chlorophyll intermediates in higher plants, but DVR proteins from different species have diverse and differing substrate preferences, although they are homologous. << Less

  Plant Physiol. 161:521-534(2013) [PubMed] [EuropePMC]