Bacterial phytoene synthase: molecular cloning,expression, and characterization of Erwinia herbicola phytoene synthase

Phytoene synthase (PSase) catalyzes the condensation of two molecules of geranylgeranyl diphosphate (GGPP) to give prephytoene diphosphate (PPPP) and the subsequent rearrangement of the cyclopropylcarbinyl intermediate to phytoene. These reactions constitute the first pathway specific step in carotenoid biosynthesis. The crtB gene encoding phytoene synthase was isolated from a plasmid containing the carotenoid gene cluster in Erwinia herbicola and cloned into an Escherichia coli expression system. Upon induction, recombinant phytoene synthase constituted 5-10% of total soluble protein. To facilitate purification of the recombinant enzyme, the structural gene for PSase was modified by site-directed mutagenesis to incorporate a C-terminal Glu-Glu-Phe (EEF) tripepetide to allow purification by immunoaffinity chromatography on an immobilized monoclonal anti-alpha-tubulin antibody YL1/2 column. Purified recombinant PSase-EEF gave a band at 34.5 kDa upon SDS-PAGE. Recombinant PSase-EEF was then purified to >90% homogeneity in two steps by ion-exchange and immunoaffinity chromatography. The enzyme required Mn(2+) for activity, had a pH optimum of 8.2, and was strongly stimulated by detergent. The concentration of GGPP needed for half-maximal activity was approximately 35 microM, and a significant inhibition of activity was seen at GGPP concentrations above 100 microM. The sole product of the reaction was 15,15'-Z-phytoene.     

Retention of triplicated phytoene synthase (PSY) genes in Brassica napus L. and its diploid progenitors during the evolution of the Brassiceae

The extent of genome redundancy exhibited by Brassica species provides a model to study the evolutionary fate of multi-copy genes and the effects of polyploidy in economically important crops. Phytoene synthase (PSY) catalyzes the first committed reaction of the carotenoid biosynthetic pathway, which has been shown to be rate-limiting in Brassica napus seeds. In Arabidopsis thaliana, a single PSY gene (AtPSY) regulates phytoene synthesis in all tissues. Considering that diploid Brassica genomes contain three Arabidopsis-like subgenomes, the objectives of the present work were to determine whether PSY gene families exist in B. napus (AACC) and its diploid progenitor species, Brassica rapa (AA) and Brassica oleracea (CC); to establish the level of retention of Brassica PSY genes; to map PSY gene family members in the A and C genomes and to compare Brassica PSY gene expression patterns. A total of 12 PSY homologues were identified, 6 in B. napus (BnaX.PSY.a-f) and 3 in B. rapa (BraA.PSY.a-c) and B. oleracea (BolC.PSY.a-c). Indeed, with six members, B. napus has the largest PSY gene family described to date. Sequence comparison between AtPSY and Brassica PSY genes revealed a highly conserved gene structure and identity percentages above 85% at the coding sequence (CDS) level. Altogether, our data indicate that PSY gene family expansion preceded the speciation of B. rapa and B. oleracea, dating back to the paralogous subgenome triplication event. In these three Brassica species, all PSY homologues are expressed, exhibiting overlapping redundancy and signs of subfunctionalization among photosynthetic and non-photosynthetic tissues. This evidence supports the hypothesis that functional divergence of PSY gene expression facilitates the accumulation of high levels of carotenoids in chromoplast-rich tissues. Thus, functional retention of triplicated Brassica PSY genes could be at least partially explained by the selective advantage provided by increased levels of gene product in floral organs. A better understanding of carotenogenesis in Brassica will aid in the future development of transgenic and conventional cultivars with carotenoid-enriched oil.     

Phylogenetic analysis and evolutionary studies of plant carotenoid cleavage dioxygenase gene

The oxidative breakdown of carotenoid evidences the formation of apocarotenoids through carotenoid cleavage dioxygenases (CCDs). Numerous CCDs and apocarotenoids have been identified and characterized in plants. Using available sequence data, a study was performed to investigate the phylogenetic relationship among CCD genes and to statistically estimate the sequence conservation and functional divergence. In total, 77 genes were identified from 39 species belonging to 21 families. Our result of phylogenetic analysis indicated the existence of well-conserved subfamilies. Moreover, comparative genomic analysis showed that the gene structures of the CCDs were highly conserved across some different lineage species. Through functional divergence analysis, a substantial divergence was found between CCD subfamilies. In addition, examination of the site-specific profile revealed the critical amino acid residues accounting for functional divergence. This study mainly focused on the evolution of CCD genes and their functional divergence which may deliver an initial step for further experimental verifications.     

Molecular evolution of two consecutive carotenoid cleavage dioxygenase genes in strigolactone biosynthesis in plants

Strigolactones are newly discovered plant hormones that perform various functions, from signaling in symbiotic interactions with arbuscular mycorrhizal fungi to controlling outgrowth of axillary buds. We examined the phylogenetic relationships of two carotenoid cleavage dioxygenase genes (CCD7 and CCD8) that are involved in consecutive upstream steps of the proposed strigolactone biosynthesis pathway. The CCD7 and CCD8 sequences from 11 model species, divided into two clades, correspond to sequences from monocotyledons and dicotyledons. However, the sequences from the primitive moss, Physcomitrella patens, appeared to be evolutionarily distinct from those of the angiosperms. CCD7 and CCD8 are much conserved, since no significant positive selection was detected among these plants. Ks values indicated that CCD7 and CCD8 diverged about 290 to 430 million years ago. As essential genes in the strigolactone pathway, the divergence timing of the conserved CCD7 and CCD8 genes reflects the approximate time of generation of strigolactone as a regulatory substance. This timing calculation also coincides with initiation of symbiosis between plants and microorganisms, inferred from the fossil record. Molecular evolution analyses of genes in metabolic pathways can provide insight concerning gene evolution.     

cDNA cloning and expression of human lactosylceramide synthase

Lactosylceramide synthase is an enzyme that catalyzes the transfer of galactose from UDP-Gal to glucosylceramide, and thus participates in the biosynthesis of most glycolipids in mammals. We have isolated and sequenced the cDNA clone encoding human lactosylceramide synthase. The deduced amino acid sequence of the human lactosylceramide synthase showed 94.2% identity with rat lactosylceramide synthase. Northern blotting analysis revealed that lactosylceramide synthase mRNA was expressed in various tissues, with the highest level in brain and adrenal gland.     

cDNA cloning and gene expression analysis of human myo-inositol 1-phosphate synthase

myo-Inositol 1-phosphate synthase (EC 5.5.1.4) (IPS) is a key enzyme in myo-inositol biosynthesis pathway. This study describes the molecular cloning of the full length human myo-inositol 1-phosphate synthase (hIPS) cDNA, tissue distribution of its mRNA and characterizes its gene expression in cultured HepG2 cells. Human testis, ovary, heart, placenta, and pancreas express relatively high level of hIPS mRNA, while blood leukocyte, thymus, skeletal muscle, and colon express low or marginal amount of the mRNA. In the presence of glucose, hIPS mRNA level increases 2- to 4-fold in HepG2 cells. hIPS mRNA is also up-regulated 2- to 3-fold by 2.5 microM lovastain. This up-regulation is prevented by mevalonic acid, farnesol, and geranylgeraniol, suggesting a G-protein mediated signal transduction mechanism in the regulation of hIPS gene expression. hIPS mRNA expression is 50% suppressed by 10mM lithium ion in these cells. Neither 5mM myo-inositol nor the three hormones: estrogen, thyroid hormone, and insulin altered hIPS mRNA expression in these cells.     

cDNA cloning and expression of human lactosylceramide synthase.

Lactosylceramide synthase is an enzyme that catalyzes the transfer of galactose from UDP-Gal to glucosylceramide, and thus participates in the biosynthesis of most glycolipids in mammals. We have isolated and sequenced the cDNA clone encoding human lactosylceramide synthase. The deduced amino acid sequence of the human lactosylceramide synthase showed 94.2% identity with rat lactosylceramide synthase. Northern blotting analysis revealed that lactosylceramide synthase mRNA was expressed in various tissues, with the highest level in brain and adrenal gland.     

On the substrate- and stereospecificity of the plant carotenoid cleavage dioxygenase 7

Strigolactones are phytohormones synthesized from carotenoids via a stereospecific pathway involving the carotenoid cleavage dioxygenases 7 (CCD7) and 8. CCD7 cleaves 9-cis-β-carotene to form a supposedly 9-cis-configured β-apo-10′-carotenal. CCD8 converts this intermediate through a combination of yet undetermined reactions into the strigolactone-like compound carlactone. Here, we investigated the substrate and stereo-specificity of the Arabidopsis and pea CCD7 and determined the stereo-configuration of the β-apo-10′-carotenal intermediate by using Nuclear Magnetic Resonance Spectroscopy. Our data unequivocally demonstrate the 9-cis-configuration of the intermediate. Both CCD7s cleave different 9-cis-carotenoids, yielding hydroxylated 9-cis-apo-10′-carotenals that may lead to hydroxylated carlactones, but show highest affinity for 9-cis-β-carotene.     

Identification and expression pattern of a new carotenoid cleavage dioxygenase gene member from Bixa orellana

Carotenoid cleavage dioxygenases (CCDs) are a class of enzymes involved in the biosynthesis of a broad diversity of secondary metabolites known as apocarotenoids. In plants, CCDs are part of a genetic family with members which cleave specific double bonds of carotenoid molecules. CCDs are involved in the production of diverse and important metabolites such as vitamin A and abscisic acid (ABA). Bixa orellana L. is the main source of the natural pigment annatto or bixin, an apocarotenoid accumulated in large quantities in its seeds. Bixin biosynthesis has been studied and the involvement of a CCD has been confirmed in vitro. However, the CCD genes involved in the biosynthesis of the wide variety of apocarotenoids found in this plant have not been well documented. In this study, a new CCD1 gene member (BoCCD1) was identified and its expression was charaterized in different plant tissues of B. orellana plantlets and adult plants. The BoCCD1 sequence showed high homology with plant CCD1s involved mainly in the cleavage of carotenoids in several sites to generate multiple apocarotenoid products. Here, the expression profiles of the BoCCD1 gene were analysed and discussed in relation to total carotenoids and other important apocarotenoids such as bixin.     

Human geranylgeranyl diphosphate synthase. cDNA cloning and expression

Geranylgeranyl diphosphate (GGPP) synthase (GGPPSase) catalyzes the synthesis of GGPP, which is an important molecule responsible for the C20-prenylated protein biosynthesis and for the regulation of a nuclear hormone receptor (LXR.RXR). The human GGPPSase cDNA encodes a protein of 300 amino acids which shows 16% sequence identity with the known human farnesyl diphosphate (FPP) synthase (FPPSase). The GGPPSase expressed in Escherichia coli catalyzes the GGPP formation (240 nmol/min/mg) from FPP and isopentenyl diphosphate. The human GGPPSase behaves as an oligomeric molecule with 280 kDa on a gel filtration column and cross-reacts with an antibody directed against bovine brain GGPPSase, which differs immunochemically from bovine brain FPPSase. Northern blot analysis indicates the presence of two forms of the mRNA.     

Vascular-specific activity of the Arabidopsis carotenoid cleavage dioxygenase 7 gene promoter

Carotenoid cleavage dioxygenases (CCDs) are involved in the production of diverse apocarotenoids including phytohormones, the visual molecules and the aromatic volatile compounds derived from carotenoids. Here, we examined the spatial expression of four of the CCD genes (AtCcd1, 4, 7 and 8) among the nine members of this family in Arabidopsis by RT-PCR. We found that the AtCcd7 gene showed strong expression in seeds. However, the promoter activity of the 1,867-bp 5′-upstream region of this gene exhibited a vascular specificity at all developmental stages throughout the transgenic Arabidopsis plants tested. The strength of the AtCcd7 promoter was also found to be lower than that of the 35S promoter by about 60%. The whole body expression of the β-glucuronidase (GUS) reporter gene driven by the AtCcd7 promoter in Arabidopsis plants was confirmed in different organs by RT-PCR and GUS enzymatic assays. Histochemical GUS staining further revealed that the AtCcd7 promoter has utility in limiting the expression of target genes to the vascular tissues in all plant organs such as the leaf, stem, root, flower and seed.