RESUMO
The enzyme geranylgeranyl diphosphate synthase (GGPS: EC 2.5.1.1, EC 2.5.1.10, EC 2.5.1.29) catalyses the formation of geranylgeranyl diphosphate (GGPP) from isopentenyl diphosphate and dimethylallyl diphosphate via three successive condensation reactions. A full-length nucleotide sequence of GGPS (named CrGGPS) was cloned from the medicinal plant Catharanthus roseus. The deduced polypeptide has 383 amino acids with a calculated mass of 41.6 kDa and possesses prenyltransferase signatures characteristic of plant type II GGPS. The enzyme was characterized by functional complementation in carotenoid accumulating strains of Escherichia coli. When cultures of Catharanthus cell lines were treated with methyljasmonate, no specific increase in transcript levels were observed. In plants, GGPS are encoded by a small multigene family and the isoforms have been shown to be localized in three different subcellular compartments: chloroplast, endoplasmic reticulum and mitochondria. We investigated the subcellular distribution of CrGGPS through transient transformations of C. roseus cells with a yellow fluorescent protein-fused construct. Our results clearly indicate that CrGGPS is located to plastids within stroma and stromules.
Assuntos
Catharanthus/enzimologia , Farnesiltranstransferase/genética , Acetatos , Sequência de Aminoácidos , Proteínas de Bactérias , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Ciclopentanos , Primers do DNA/genética , DNA Complementar/biossíntese , Escherichia coli , Farnesiltranstransferase/metabolismo , Teste de Complementação Genética , Espaço Intracelular/metabolismo , Proteínas Luminescentes , Microscopia de Fluorescência , Dados de Sequência Molecular , Oxilipinas , Plastídeos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNARESUMO
Farnesyl diphosphate (FPP) synthase (FPS: EC.2.5.1.1, EC.2.5.1.10) catalyzes the formation of FPP from isopentenyl diphosphate and dimethylallyl diphosphate via two successive condensation reactions. A cDNA designated CrFPS, encoding a protein showing high similarities with trans-type short FPS isoforms, was isolated from the Madagascar periwinkle (Catharanthus roseus). This cDNA was shown to functionally complement the lethal FPS deletion mutant in the yeast Saccharomyces cerevisiae. At the subcellular level, while short FPS isoforms are usually described as cytosolic proteins, we showed, using transient transformations of C. roseus cells with yellow fluorescent protein-fused constructs, that CrFPS is targeted to peroxisomes. This finding is discussed in relation to the subcellular distribution of FPS isoforms in plants and animals and opens new perspectives towards the understanding of isoprenoid biosynthesis.
Assuntos
Catharanthus/enzimologia , Geraniltranstransferase/metabolismo , Peroxissomos/metabolismo , Terpenos/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias , Sequência de Bases , Transporte Biológico , Catharanthus/genética , Catharanthus/fisiologia , Clonagem Molecular , DNA Complementar/genética , Teste de Complementação Genética , Geraniltranstransferase/química , Geraniltranstransferase/genética , Hemiterpenos/metabolismo , Proteínas Luminescentes , Dados de Sequência Molecular , Compostos Organofosforados/metabolismo , RNA de Plantas/genética , RNA de Plantas/isolamento & purificação , Proteínas Recombinantes de Fusão , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Análise de Sequência de DNA , Deleção de SequênciaRESUMO
In plants, the mevalonic acid (MVA) pathway provides precursors for the formation of triterpenes, sesquiterpenes, phytosterols and primary metabolites important for cell integrity. Here, we have cloned the cDNA encoding enzymes catalysing the final three steps of the MVA pathway from Madagascar periwinkle (Catharanthus roseus), mevalonate kinase (MVK), 5-phosphomevalonate kinase (PMK) and mevalonate 5-diphosphate decarboxylase (MVD). These cDNA were shown to functionally complement MVA pathway deletion mutants in the yeast Saccharomyces cerevisiae. Transient transformations of C. roseus cells with yellow fluorescent protein (YFP)-fused constructs reveal that PMK and MVD are localised to the peroxisomes, while MVK was cytosolic. These compartmentalisation results were confirmed using the Arabidopsis thaliana MVK, PMK and MVD sequences fused to YFP. Based on these observations and the arguments raised here we conclude that the final steps of the plant MVA pathway are localised to the peroxisome.
