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Gerhard Sandmann - One of the best experts on this subject based on the ideXlab platform.
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Gene sll0033 from Synechocystis 6803 encodes a Carotene isomerase involved in the biosynthesis of all-E lycopene.
Zeitschrift für Naturforschung C, 2001Co-Authors: Jürgen Breitenbach, Agustín Vioque, Gerhard SandmannAbstract:: The function of gene sll0033 from Synechocystis 6803 which is homologous to the bacterial crtI-type phytoene desaturase genes was elucidated as a novel Carotene isomerase. Escherichia coli transformed with all genes necessary for the formation of Zeta-Carotene and expressing a Zeta-Carotene desaturase synthesized the positional isomer prolycopene (7,9,7',9'Z lycopene) which cannot be cyclized in the subsequent reactions to a- and beta-Carotene. Upon cotransformation with sll0033, the formation of all-E lycopene is mediated instead.
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catalytic properties of an expressed and purified higher plant type Zeta Carotene desaturase from capsicum annuum
FEBS Journal, 1999Co-Authors: Jürgen Breitenbach, Shinichi Takaichi, Marcel Kuntz, Gerhard SandmannAbstract:The ζ-Carotene desaturase from Capsicum annuum (EC 1.14.99.-) was expressed in Escherichia coli, purified and characterized biochemically. The enzyme acts as a monomer with lipophilic quinones as cofactors. Km values for the substrate ζ-Carotene or the intermediate neurosporene in the two-step desaturation reaction are almost identical. Product analysis showed that different lycopene isomers are formed, including substantial amounts of the all-trans form, together with 7,7′,9,9′-tetracis prolycopene via the corresponding neurosporene isomers. The application of different geometric isomers as substrates revealed that the ζ-Carotene desaturase has no preference for certain isomers and that the nature of the isomers formed during catalysis depends strictly on the isomeric composition of the substrate.
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a higher plant type Zeta Carotene desaturase in the cyanobacterium synechocystis pcc6803
Plant Molecular Biology, 1998Co-Authors: Jürgen Breitenbach, Agustín Vioque, Blanca Fernandezgonzalez, Gerhard SandmannAbstract:The genomic DNA sequence of Synechocystis was analysed for putative ζ-Carotene desaturase genes. Two promising candidates slr0940 and slr0033 were found with similarities to the structurally different ζ-Carotene desaturase genes from higher plants and Anabaena, respectively. Only the expression product of the analogue to the plant gene, slr0940, was able to mediate the 2-step desaturation of ζ-Carotene via neurosporene to lycopene after complementation of this pathway in Escherichia coli. When enzyme reactions were carried out with this protein, activity was obtained with either ζ-Carotene or neuroporene as substrates. The in vitro reaction was inhibited by the pyrimidine derivative J852 which is effective as experimental herbicide in plants. The occurrence of two different types of ζ-Carotene desaturases among cyanobacteria and the phylogenetic consequences on chloroplast evolution are discussed.
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A new non-radioactive assay of phytoene desaturase to evaluate bleaching herbicides
Zeitschrift für Naturforschung. C. A journal of biosciences, 1996Co-Authors: Gerhard Sandmann, Christian Schneider, Peter BögerAbstract:: A non-radioactive cell-free assay was developed to quantitatively determine inhibition of plant-type phytoene desaturase by bleaching herbicides. An active desaturase was prepared from an appropriately cloned E. coli transformant. Another E. coli transformant was used to produce the required phytoene. Phytofluene and Zeta-Carotene, the products of the desaturase reaction, were either determined by HPLC or optical absorption spectra. Enzyme kinetics and inhibition data for the bleaching tetrazole herbicide WL110547 are presented as an example.
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Purification in an active state and properties of the 3-step phytoene desaturase from Rhodobacter capsulatus overexpressed in Escherichia coli.
Journal of Biochemistry, 1996Co-Authors: Axel Raisig, Glenn E. Bartley, Pablo A. Scolnik, Gerhard SandmannAbstract:: The phytoene desaturase gene from Rhodobacter capsulatus was expressed in Escherichia coli and the resulting protein was purified. The purification steps involved were ammonium sulfate precipitation and ion exchange chromatography, leading to a homogenous protein of 57 kDa with high specific enzymatic activity. The purified enzyme was characterized with respect to substrate specificity and product formation. In addition to phytoene, the intermediates, phytofluene and Zeta-Carotene, were both converted to neurosporene, the end product of the reaction. Furthermore, 1,2-epoxy phytoene was a suitable substrate whereas the C30 diapophytoene was not. The Km values for phytoene and Zeta-Carotene were determined to be 33.3 and 16.6 microM, respectively. The desaturation reaction is dependent on the cofactor FAD. Oxidized nicotine nucleotides or ATP had no positive effect. The Km value for FAD was 4.9 microM. Inhibition of the desaturation reaction was observed with diphenylamine.
Jürgen Breitenbach - One of the best experts on this subject based on the ideXlab platform.
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Gene sll0033 from Synechocystis 6803 encodes a Carotene isomerase involved in the biosynthesis of all-E lycopene.
Zeitschrift für Naturforschung C, 2001Co-Authors: Jürgen Breitenbach, Agustín Vioque, Gerhard SandmannAbstract:: The function of gene sll0033 from Synechocystis 6803 which is homologous to the bacterial crtI-type phytoene desaturase genes was elucidated as a novel Carotene isomerase. Escherichia coli transformed with all genes necessary for the formation of Zeta-Carotene and expressing a Zeta-Carotene desaturase synthesized the positional isomer prolycopene (7,9,7',9'Z lycopene) which cannot be cyclized in the subsequent reactions to a- and beta-Carotene. Upon cotransformation with sll0033, the formation of all-E lycopene is mediated instead.
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catalytic properties of an expressed and purified higher plant type Zeta Carotene desaturase from capsicum annuum
FEBS Journal, 1999Co-Authors: Jürgen Breitenbach, Shinichi Takaichi, Marcel Kuntz, Gerhard SandmannAbstract:The ζ-Carotene desaturase from Capsicum annuum (EC 1.14.99.-) was expressed in Escherichia coli, purified and characterized biochemically. The enzyme acts as a monomer with lipophilic quinones as cofactors. Km values for the substrate ζ-Carotene or the intermediate neurosporene in the two-step desaturation reaction are almost identical. Product analysis showed that different lycopene isomers are formed, including substantial amounts of the all-trans form, together with 7,7′,9,9′-tetracis prolycopene via the corresponding neurosporene isomers. The application of different geometric isomers as substrates revealed that the ζ-Carotene desaturase has no preference for certain isomers and that the nature of the isomers formed during catalysis depends strictly on the isomeric composition of the substrate.
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a higher plant type Zeta Carotene desaturase in the cyanobacterium synechocystis pcc6803
Plant Molecular Biology, 1998Co-Authors: Jürgen Breitenbach, Agustín Vioque, Blanca Fernandezgonzalez, Gerhard SandmannAbstract:The genomic DNA sequence of Synechocystis was analysed for putative ζ-Carotene desaturase genes. Two promising candidates slr0940 and slr0033 were found with similarities to the structurally different ζ-Carotene desaturase genes from higher plants and Anabaena, respectively. Only the expression product of the analogue to the plant gene, slr0940, was able to mediate the 2-step desaturation of ζ-Carotene via neurosporene to lycopene after complementation of this pathway in Escherichia coli. When enzyme reactions were carried out with this protein, activity was obtained with either ζ-Carotene or neuroporene as substrates. The in vitro reaction was inhibited by the pyrimidine derivative J852 which is effective as experimental herbicide in plants. The occurrence of two different types of ζ-Carotene desaturases among cyanobacteria and the phylogenetic consequences on chloroplast evolution are discussed.
Joseph Hirschberg - One of the best experts on this subject based on the ideXlab platform.
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A mutant allele of Zeta-Carotene isomerase (Z-ISO) is associated with the yellow pigmentation of the ‘Pinalate’ sweet orange mutant and reveals insights into its role in fruit carotenogenesis
2019Co-Authors: María-jesús Rodrigo, Joseph Hirschberg, Joanna Lado, Enriqueta Alos, Berta Alquezar, Orly Dery, Lorenzo ZacaríasAbstract:Abstract Fruit coloration is one of the main quality parameters of Citrus fruit primarily determined by genetic factors. The fruit of ordinary sweet orange (Citrus sinensis) displays a pleasant orange tint due to the accumulation carotenoids, representing b,b-xanthophylls more than 80% of the total content. Pinalate is a spontaneous bud mutant derived from sweet orange Navelate, characterized by yellow fruits due to elevated proportions of upstream Carotenes and reduced b,b-xanthophylls. To identify the molecular basis of Pinalate yellow fruit, a complete characterization of carotenoids profile together with transcriptional changes in carotenoid biosynthetic genes were performed in mutant and parental fruits during development and ripening. Pinalate fruit tissues showed a distinctive carotenoid profile at all ripening stages, accumulating phytoene, phytofluene and unusual proportions of 9,15,9´-tri-cis- and 9,9´-di-cis-z-Carotene, while content of downstream carotenoids was significantly decreased. Transcript levels for most of the carotenoid biosynthetic genes showed no alterations in Pinalate; however, the steady-state level mRNA of z-Carotene isomerase (Z-ISO), which catalyses the conversion of 9,15,9´-tri-cis- to 9,9´-di-cis-z-Carotene, was significantly reduced in Pinalate fruit and leaf tissues. The isolation of the Pinalate Z-ISO genomic sequence identified a new allele with a single nucleotide insertion at the second exon, which generates an alternative splicing site that alters Z-ISO transcripts encoding non-functional enzyme. Moreover, functional assays of citrus Z-ISO in E.coli showed that light is able to enhance a non-enzymatic isomerization of tri-cis to di-cis-z-Carotene which is in agreement with the partial rescue of mutant phenotype when Pinalate fruits are highly exposed to light during ripening. The defect in Pinalate Z-ISO gene causes a bottleneck in the carotenoid pathway with an unbalanced content of Carotenes upstream to b,b-xanthophylls in fruit tissues. Taken together, our results indicate that a spontaneous single nucleotide insertion in Z-ISO is the molecular basis of the altered pigmentation in Pinalate sweet orange mutant and points this isomerase as an essential activity for carotenogenesis in citrus fruits.
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Molecular structure and enzymatic function of lycopene cyclase from the cyanobacterium Synechococcus sp strain PCC7942.
The Plant Cell, 1994Co-Authors: Francis X. Cunningham, Daniel Chamovitz, Joseph Hirschberg, Elisabeth GanttAbstract:A gene encoding the enzyme lycopene cyclase in the cyanobacterium Synechococcus sp strain PCC7942 was mapped by genetic complementation, cloned, and sequenced. This gene, which we have named crtL, was expressed in strains of Escherichia coli that were genetically engineered to accumulate the carotenoid precursors lycopene, neurosporene, and Zeta-Carotene. The crtL gene product converts the acyclic hydrocarbon lycopene into the bicyclic beta-Carotene, an essential component of the photosynthetic apparatus in oxygen-evolving organisms and a source of vitamin A in human and animal nutrition. The enzyme also converts neurosporene to the monocyclic beta-zeaCarotene but does not cyclize Zeta-Carotene, indicating that desaturation of the 7-8 or 79-89 carbon-carbon bond is required for cyclization. The bleaching herbicide 2-(4-methylphenoxy)triethylamine hydrochloride (MPTA) effectively inhibits both cyclization reactions. A mutation that confers resistance to MPTA in Synechococcus sp PCC7942 was identified as a point mutation in the promoter region of crtL. The deduced amino acid sequence of lycopene cyclase specifies a polypeptide of 411 amino acids with a molecular weight of 46,125 and a pI of 6.0. An amino acid sequence motif indicative of FAD utilization is located at the N terminus of the polypeptide. DNA gel blot hybridization analysis indicated a single copy of crtL in Synechococcus sp PCC7942. Other than the FAD binding motif, the predicted amino acid sequence of the cyanobacterial lycopene cyclase bears little resemblance to the two known lycopene cyclase enzymes from nonphotosynthetic bacteria. Preliminary results from DNA gel blot hybridization experiments suggest that, like two earlier genes in the pathway, the Synechococcus gene encoding lycopene cyclase is homologous to plant and algal genes encoding this enzyme.
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a single polypeptide catalyzing the conversion of phytoene to Zeta Carotene is transcriptionally regulated during tomato fruit ripening
Proceedings of the National Academy of Sciences of the United States of America, 1992Co-Authors: Iris Pecker, Daniel Chamovitz, Hartmut Linden, Gerhard Sandmann, Joseph HirschbergAbstract:The cDNA of the gene pds from tomato, encoding the carotenoid biosynthesis enzyme phytoene desaturase, was cloned, and its nucleotide sequence was determined. Cells of Escherichia coli that expressed the tomato pds gene could convert phytoene to Zeta-Carotene. This result suggests that one polypeptide, the product of the pds gene, can carry out phytoene desaturation in the carotenoid biosynthetic pathway. Transcripts of the pds gene accumulate in orange tomato fruit, indicating transcriptional control of pds expression during fruit ripening. The deduced amino acid sequence of phytoene desaturase indicates that this enzyme in tomato contains 583 amino acids that are highly conserved with respect to the homologous enzymes in cyanobacteria and algae. The deduced amino acid sequences of the phytoene desaturases from other microorganisms (purple bacteria and fungi) appear to be evolutionarily unrelated to those from green photosynthetic organisms.
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Functional complementation in Escherichia coli of different phytoene desaturase genes and analysis of accumulated Carotenes.
Zeitschrift für Naturforschung C, 1991Co-Authors: Hartmut Linden, Iris Pecker, Daniel Chamovitz, Joseph Hirschberg, Norihiko Misawa, Gerhard SandmannAbstract:: Three different phytoene desaturase genes, from Rhodobacter capsulatus, Erwinia uredovora, and Synechococcus PCC 7942, have been functionally complemented with a gene construct from E. uredovora which encodes all enzymes responsible for formation of 15-cis phytoene in Escherichia coli. As indicated by the contrasting reaction products detected in the pigmented E. coli cells after co-transformation, a wide functional diversity of these three different types of phytoene desaturases can be concluded. The Carotenes formed by the phytoene desaturase from R. capsulatus were trans-neurosporene with three additional double bonds and two cis isomers. Furthermore, small amounts of three Zeta-Carotene isomers (2 double bonds more than phytoene) and phytofluene (15-cis and all-trans with + 1 double bond) were detected as intermediates. When the subsequent genes from E. uredovora which encode for lycopene cyclase and beta-Carotene hydroxylase were present, neurosporene, the phytoene desaturase product of R. capsulatus, was subsequently converted to the monocyclic beta-zeaCarotene and its monohydroxylation product. The most abundant Carotene resulting from phytoene desaturation by the E. uredovora enzyme was trans-lycopene together with a cis isomer. In addition, bisdehydrolycopene was also formed. The reaction products of Synechococcus phytoene desaturase were two cis isomers of Zeta-Carotene and only small amounts of trans-Zeta-Carotene including 15-cis. The I50 values for flurtamone and diphenylamine to inhibit phytoene desaturation were determined and differential inhibition was observed for diphenylamine.
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Molecular cloning and expression in photosynthetic bacteria of a soybean cDNA coding for phytoene desaturase, an enzyme of the carotenoid biosynthesis pathway.
Proceedings of the National Academy of Sciences of the United States of America, 1991Co-Authors: Glenn E. Bartley, Iris Pecker, Daniel Chamovitz, Joseph Hirschberg, Paul V. Viitanen, Pablo A. ScolnikAbstract:Carotenoids are orange, yellow, or red photo-protective pigments present in all plastids. The first carotenoid of the pathway is phytoene, a colorless compound that is converted into colored carotenoids through a series of desaturation reactions. Genes coding for carotenoid desaturases have been cloned from microbes but not from plants. We report the cloning of a cDNA for pds1, a soybean (Glycine max) gene that, based on a complementation assay using the photosynthetic bacterium Rhodobacter capsulatus, codes for an enzyme that catalyzes the two desaturation reactions that convert phytoene into Zeta-Carotene, a yellow carotenoid. The 2281-base-pair cDNA clone analyzed contains an open reading frame with the capacity to code for a 572-residue protein of predicted Mr 63,851. Alignment of the deduced Pds1 peptide sequence with the sequences of fungal and bacterial carotenoid desaturases revealed conservation of several amino acid residues, including a dinucleotide-binding motif that could mediate binding to FAD. The Pds1 protein is synthesized in vitro as a precursor that, upon import into isolated chloroplasts, is processed to a smaller mature form. Hybridization of the pds1 cDNA to genomic blots indicated that this gene is a member of a low-copy-number gene family. One of these loci was genetically mapped using restriction fragment length polymorphisms between Glycine max and Glycine soja. We conclude that pds1 is a nuclear gene encoding a phytoene desaturase enzyme that, as its microbial counterparts, contains sequence motifs characteristic of flavoproteins.
Guoying Wang - One of the best experts on this subject based on the ideXlab platform.
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mutations in the maize Zeta Carotene desaturase gene lead to viviparous kernel
PLOS ONE, 2017Co-Authors: Yan Chen, Jiankun Li, Yicong Du, Jing Xu, Jun Zheng, Junjie Fu, Guoying WangAbstract:Preharvest sprouting reduces the maize quality and causes a significant yield loss in maize production. vp-wl2 is a Mutator (Mu)-induced viviparous mutant in maize, causing white or pale yellow kernels, dramatically reduced carotenoid and ABA content, and a high level of Zeta-Carotene accumulation. Here, we reported the cloning of the vp-wl2 gene using a modified digestion-ligation-amplification method (DLA). The results showed that an insertion of Mu9 in the first intron of the Zeta-Carotene desaturase (ZDS) gene results in the vp-wl2 mutation. Previous studies have suggested that ZDS is likely the structural gene of the viviparous9 (vp9) locus. Therefore, we performed an allelic test using vp-wl2 and three vp9 mutants. The results showed that vp-wl2 is a novel allele of the vp9 locus. In addition, the sequences of ZDS gene were identified in these three vp9 alleles. The vp-wl2 mutant gene was subsequently introgressed into four maize inbred lines, and a viviparous phenotype was observed with yield losses from 7.69% to 13.33%.
Hartmut Linden - One of the best experts on this subject based on the ideXlab platform.
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biochemical characterization of purified Zeta Carotene desaturase from anabaena pcc 7120 after expression in escherichia coli
FEBS Journal, 1996Co-Authors: Manuela Albrecht, Hartmut Linden, Gerhard SandmannAbstract:A novel enzyme, ζ-Carotene desaturase from the cyanobacterium Anabaena, which catalyzes the last two steps in a series of desaturations, was overexpressed in Escherichia coli. For the first time, this allowed the purification of this enzyme and subsequent enzyme kinetic studies. The enzyme was solubilized from the E. coli membranes by Chaps and purified to homogeneity by ammonium sulfate precipitation, ion-exchange and hydrophobic interaction chromatography. The correct translational start was confirmed by N-terminal protein sequencing. Substrates for ζ-Carotene desaturase apart from ζ-Carotene are those Carotenes which partially resemble the latter, like neurosporene and β-zeaCarotene yielding lycopene and γ-Carotene, respectively as reaction products. Also cis isomers like pro-ζ-Carotene were converted to the correspondiong products. Km values of 10 μM were determined for both substrates ζ-Carotene and neurosporene. The enzyme was inhibited to some extent by the experimental herbicides J852 and LS80707 and also by diphenylamine which is a well-known inhibitor of the bacterial-type phytoene desaturase.
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a single polypeptide catalyzing the conversion of phytoene to Zeta Carotene is transcriptionally regulated during tomato fruit ripening
Proceedings of the National Academy of Sciences of the United States of America, 1992Co-Authors: Iris Pecker, Daniel Chamovitz, Hartmut Linden, Gerhard Sandmann, Joseph HirschbergAbstract:The cDNA of the gene pds from tomato, encoding the carotenoid biosynthesis enzyme phytoene desaturase, was cloned, and its nucleotide sequence was determined. Cells of Escherichia coli that expressed the tomato pds gene could convert phytoene to Zeta-Carotene. This result suggests that one polypeptide, the product of the pds gene, can carry out phytoene desaturation in the carotenoid biosynthetic pathway. Transcripts of the pds gene accumulate in orange tomato fruit, indicating transcriptional control of pds expression during fruit ripening. The deduced amino acid sequence of phytoene desaturase indicates that this enzyme in tomato contains 583 amino acids that are highly conserved with respect to the homologous enzymes in cyanobacteria and algae. The deduced amino acid sequences of the phytoene desaturases from other microorganisms (purple bacteria and fungi) appear to be evolutionarily unrelated to those from green photosynthetic organisms.
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Functional complementation in Escherichia coli of different phytoene desaturase genes and analysis of accumulated Carotenes.
Zeitschrift für Naturforschung C, 1991Co-Authors: Hartmut Linden, Iris Pecker, Daniel Chamovitz, Joseph Hirschberg, Norihiko Misawa, Gerhard SandmannAbstract:: Three different phytoene desaturase genes, from Rhodobacter capsulatus, Erwinia uredovora, and Synechococcus PCC 7942, have been functionally complemented with a gene construct from E. uredovora which encodes all enzymes responsible for formation of 15-cis phytoene in Escherichia coli. As indicated by the contrasting reaction products detected in the pigmented E. coli cells after co-transformation, a wide functional diversity of these three different types of phytoene desaturases can be concluded. The Carotenes formed by the phytoene desaturase from R. capsulatus were trans-neurosporene with three additional double bonds and two cis isomers. Furthermore, small amounts of three Zeta-Carotene isomers (2 double bonds more than phytoene) and phytofluene (15-cis and all-trans with + 1 double bond) were detected as intermediates. When the subsequent genes from E. uredovora which encode for lycopene cyclase and beta-Carotene hydroxylase were present, neurosporene, the phytoene desaturase product of R. capsulatus, was subsequently converted to the monocyclic beta-zeaCarotene and its monohydroxylation product. The most abundant Carotene resulting from phytoene desaturation by the E. uredovora enzyme was trans-lycopene together with a cis isomer. In addition, bisdehydrolycopene was also formed. The reaction products of Synechococcus phytoene desaturase were two cis isomers of Zeta-Carotene and only small amounts of trans-Zeta-Carotene including 15-cis. The I50 values for flurtamone and diphenylamine to inhibit phytoene desaturation were determined and differential inhibition was observed for diphenylamine.