Molecular cloning and functional expression of geranylgeranyl pyrophosphate synthase from Coleus forskohlii Briq

BACKGROUND: Isopentenyl diphosphate (IPP), a common biosynthetic precursor to the labdane diterpene forskolin, has been biosynthesised via a non-mevalonate pathway. Geranylgeranyl diphosphate (GGPP) synthase is an important branch point enzyme in terpenoid biosynthesis. Therefore, GGPP synthase is thought to be a key enzyme in biosynthesis of forskolin. Herein we report the first confirmation of the GGPP synthase gene in Coleus forskohlii Briq. RESULTS: The open reading frame for full-length GGPP synthase encodes a protein of 359 amino acids, in which 1,077 nucleotides long with calculated molecular mass of 39.3 kDa. Alignments of C. forskohlii GGPP synthase amino acid sequences revealed high homologies with other plant GGPP synthases. Several highly conserved regions, including two aspartate-rich motifs were identified. Transient expression of the N-terminal region of C. forskohlii GGPP synthase-GFP fusion protein in tobacco cells demonstrated subcellular localization in the chloroplast. Carotenoid production was observed in Escherichia coli harboring pACCAR25ΔcrtE from Erwinia uredovora and plasmid carrying C. forskohlii GGPP synthase. These results suggested that cDNA encoded functional GGPP synthase. Furthermore, C. forskohlii GGPP synthase expression was strong in leaves, decreased in stems and very little expression was observed in roots. CONCLUSION: This investigation proposed that forskolin was synthesised via a non-mevalonate pathway. GGPP synthase is thought to be involved in the biosynthesis of forskolin, which is primarily synthesised in the leaves and subsequently accumulates in the stems and roots

Histone Chaperone Asf1 Plays an Essential Role in Maintaining Genomic Stability in Fission Yeast

The histone H3-H4 chaperone Asf1 is involved in chromatin assembly (or disassembly), histone exchange, regulation of transcription, and chromatin silencing in several organisms. To investigate the essential functions of Asf1 in Schizosaccharomyces pombe, asf1-ts mutants were constructed by random mutagenesis using PCR. One mutant (asf1-33(ts)) was mated with mutants in 77 different kinase genes to identify synthetic lethal combinations. The asf1-33 mutant required the DNA damage checkpoint factors Chk1 and Rad3 for its survival at the restrictive temperature. Chk1, but not Cds1, was phosphorylated in the asf1-33 mutant at the restrictive temperature, indicating that the DNA damage checkpoint was activated in the asf1-33 mutant. DNA damage occured in the asf1-33 mutant, with degradation of the chromosomal DNA observed through pulse-field gel electrophoresis and the formation of Rad22 foci. Sensitivity to micrococcal nuclease in the asf1-33 mutant was increased compared to the asf1+ strain at the restrictive temperature, suggesting that asf1 mutations also caused a defect in overall chromatin structure. The Asf1-33 mutant protein was mislocalized and incapable of binding histones. Furthermore, histone H3 levels at the centromeric outer repeat region were decreased in the asf1-33 mutant and heterochromatin structure was impaired. Finally, sim3, which encodes a CenH3 histone chaperone, was identified as a strong suppressor of the asf1-33 mutant. Taken together, these results clearly indicate that Asf1 plays an essential role in maintaining genomic stability in S. pombe

Molecular cloning and sequencing of the glycogen phosphorylase gene from Escherichia coli

AbstractThe glgP gene, which codes for glycogen phosphorylase, was cloned from a genomic library of Escherichia coli. The nucleotide sequence of the glgP gene contained a single open reading frame encoding a protein consisting of 790 amino acid residues. The glgP gene product, a polypeptide of Mr 87 000, was confirmed by SDS-polyacrylamide gel electrophoresis. The deduced amino acid sequence showed that homology between glgP of E. coli and rabbit glgP, human glgP, potato glgP, and E. coli malP was 48.6, 48.6, 42.3, and 46.1%, respectively. Within this homologous region, the active site, glycogen storage site, and pyridoxal-5′-phosphate binding site are well conserved. The enzyme activity of glycogen phosphorylase increased after introduction on a multicopy of the glgP gene

A solanesyl-diphosphate synthase localizes in glycosomes of Trypanosoma cruzi

Fil: Ferella, Marcela. ANLIS Dr. C. G. Malbrán. Instituto Nacional de Parasitología "Dr. M. Fatala Chabén" (INP); Argentina.Fil: Montalvetti, Andrea. University of Illinois. Department of Pathobiology; Estados Unidos.Fil: Rohloff, Peter. University of Illinois. Department of Pathobiology; Estados Unidos.Fil: Miranda, Kildare. University of Georgia. Center for Tropical and Emerging Global Diseases. Department of Cellular Biology; Estados Unidos.Fil: Fang, Jianmin. University of Georgia. Center for Tropical and Emerging Global Diseases. Department of Cellular Biology; Estados Unidos.Fil: Reina, Silvia. ANLIS Dr. C. G. Malbrán. Instituto Nacional de Parasitología "Dr. M. Fatala Chabén" (INP); Argentina.Fil: Kawamukai, Makoto. University Matsue. Faculty of Life and Environmental Science. Department of Applied Bioscience and Biotechnology; Japón.Fil: Bua, Jacqueline. ANLIS Dr. C. G. Malbrán. Instituto Nacional de Parasitología "Dr. M. Fatala Chabén" (INP); Argentina.Fil: Nilsson, Daniel. Karolinska Institute. Center for Genomics and Bioinformatics; Suecia.Fil: Pravia, Carlos. ANLIS Dr. C. G. Malbrán. Instituto Nacional de Parasitología "Dr. M. Fatala Chabén" (INP); Argentina.Fil: Katzin, Alejandro. Universidade de Sao Paulo. Instituto de Ciencias Biomédicas. Departamento de Parasitologia; Brasil.Fil: Casera, María B. Universidade de Sao Paulo. Instituto de Ciencias Biomédicas. Departamento de Parasitologia; Brasil.Fil: Áslund, Lena. Uppsala University. Department of Genetics and Pathology; Suecia.Fil: Andersson, Björn. Karolinska Institute. Center for Genomics and Bioinformatics; Suecia.Fil: Docampo, Roberto. University of Illinois. Department of Pathobiology; Estados Unidos.Fil: Bontempi, Esteban. ANLIS Dr. C. G. Malbrán. Instituto Nacional de Parasitología "Dr. M. Fatala Chabén"; Argentina.We report the cloning of a Trypanosoma cruzi gene encoding a solanesyl-diphosphate synthase, TcSPPS. The amino acid sequence (molecular mass ∼ 39 kDa) is homologous to polyprenyl-diphosphate synthases from different organisms, showing the seven conserved motifs and the typical hydrophobic profile. TcSPPS preferred geranylgeranyl diphosphate as the allylic substrate. The final product, as determined by TLC, had nine isoprene units. This suggests that the parasite synthesizes mainly ubiquinone-9 (UQ-9), as described for Trypanosoma brucei and Leishmania major. In fact, that was the length of the ubiquinone extracted from epimastigotes, as determined by high-performance liquid chromatography. Expression of TcSPPS was able to complement an Escherichia coli ispB mutant. A punctuated pattern in the cytoplasm of the parasite was detected by immunofluorescence analysis with a specific polyclonal antibody against TcSPPS. An overlapping fluorescence pattern was observed using an antibody directed against the glycosomal marker pyruvate phosphate dikinase, suggesting that this step of the isoprenoid biosynthetic pathway is located in the glycosomes. Co-localization in glycosomes was confirmed by immunogold electron microscopy and subcellular fractionation. Because UQ has a central role in energy production and in reoxidation of reduction equivalents, TcSPPS is promising as a new chemotherapeutic target

A subunit of decaprenyl diphosphate synthase stabilizes octaprenyl diphosphate synthase in Escherichia coli by forming a high-molecular weight complex

AbstractThe length of the isoprenoid-side chain in ubiquinone, an essential component of the electron transport chain, is defined by poly-prenyl diphosphate synthase, which comprises either homomers (e.g., IspB in Escherichia coli) or heteromers (e.g., decaprenyl diphosphate synthase (Dps1) and D-less polyprenyl diphosphate synthase (Dlp1) in Schizosaccharomyces pombe and in humans). We found that expression of either dlp1 or dps1 recovered the thermo-sensitive growth of an E. coli ispBR321A mutant and restored IspB activity and production of Coenzyme Q-8. IspB interacted with Dlp1 (or Dps1), forming a high-molecular weight complex that stabilized IspB, leading to full functionality.Structured summary:MINT-7385426:Dlp1 (uniprotkb:Q86YH6) and IspB (uniprotkb:P0AD57) physically interact (MI:0915) by blue native page (MI:0276)MINT-7385083, MINT-7385058:IspB (uniprotkb:P0AD57) and IspB (uniprotkb:P0AD57) bind (MI:0407) by blue native page (MI:0276)MINT-7385413:Dlp1 (uniprotkb:O13851) and IspB (uniprotkb:P0AD57) physically interact (MI:0915) by blue native page (MI:0276)MINT-7385024:IspB (uniprotkb:P0AD57) physically interacts (MI:0915) with Dps1 (uniprotkb:O43091) by pull down (MI:0096)MINT-7385041:IspB (uniprotkb:P0AD57) physically interacts (MI:0915) with Dlp1 (uniprotkb:O13851) by pull down (MI:0096)MINT-7385388:IspB (uniprotkb:P0AD57) and Dps1 (uniprotkb:O43091) physically interact (MI:0915) by blue native page (MI:0276

cAMP-dependent protein kinase involves calcium tolerance through the regulation of Prz1 in <i>Schizosaccharomyces pombe</i>

<p>The cAMP-dependent protein kinase Pka1 is known as a regulator of glycogenesis, meiosis, and stress responses in <i>Schizosaccharomyces pombe</i>. We demonstrated that Pka1 is responsible for calcium tolerance. Loss of functional components of the PKA pathway such as Git3, Gpa2, Cyr1, and Pka1 yields a CaCl₂-sensitive phenotype, while loss of Cgs1, a regulatory subunit of PKA, results in CaCl₂ tolerance. Cytoplasmic distribution of Cgs1 and Pka1 is increased by the addition of CaCl₂, suggesting that CaCl₂ induces dissociation of Cgs1 and Pka1. The expression of Prz1, a transcriptional regulator in calcium homeostasis, is elevated in a <i>pka1∆</i> strain and in a wild type strain under glucose-limited conditions. Accordingly, higher expression of Prz1 in the wild type strain results in a CaCl₂-sensitive phenotype. These findings suggest that Pka1 is essential for tolerance to exogenous CaCl₂, probably because the expression level of Prz1 needs to be properly regulated by Pka1.</p