Overexpression of an Archaeal Geranylgeranyl Diphosphate Synthase in<i>Escherichia coli</i>Cells

Ohto, Chikara; Nakane, Hiroyuki; Hemmi, Hisashi; Ohnuma, Shin-ichi; Obata, Seiji; Nishino, Tokuzo

doi:10.1271/bbb.62.1243

Cited by 16 publications

(12 citation statements)

References 11 publications

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“…An active form of GGPPS from Thermus thermophilus and Sulfolobus acidocaldarius was also overexpressed in E. coli cells as a GST fusion protein. Ohto et al suggested that the presence of the GST-tag leads to thermal stability of the recombinant enzymes [36]. …”

Section: Discussionmentioning

confidence: 99%

Cloning and characterization of bifunctional enzyme farnesyl diphosphate/geranylgeranyl diphosphate synthase from Plasmodium falciparum

Jordão

Gabriel

Alves

et al. 2013

Malar J

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BackgroundIsoprenoids are the most diverse and abundant group of natural products. In Plasmodium falciparum, isoprenoid synthesis proceeds through the methyl erythritol diphosphate pathway and the products are further metabolized by farnesyl diphosphate synthase (FPPS), turning this enzyme into a key branch point of the isoprenoid synthesis. Changes in FPPS activity could alter the flux of isoprenoid compounds downstream of FPPS and, hence, play a central role in the regulation of a number of essential functions in Plasmodium parasites.MethodsThe isolation and cloning of gene PF3D7_18400 was done by amplification from cDNA from mixed stage parasites of P. falciparum. After sequencing, the fragment was subcloned in pGEX2T for recombinant protein expression. To verify if the PF3D7_1128400 gene encodes a functional rPfFPPS protein, its catalytic activity was assessed using the substrate [4-14C] isopentenyl diphosphate and three different allylic substrates: dimethylallyl diphosphate, geranyl diphosphate or farnesyl diphosphate. The reaction products were identified by thin layer chromatography and reverse phase high-performance liquid chromatography. To confirm the product spectrum formed of rPfFPPS, isoprenic compounds were also identified by mass spectrometry. Apparent kinetic constants KM and Vmax for each substrate were determined by Michaelis–Menten; also, inhibition assays were performed using risedronate.ResultsThe expressed protein of P. falciparum FPPS (rPfFPPS) catalyzes the synthesis of farnesyl diphosphate, as well as geranylgeranyl diphosphate, being therefore a bifunctional FPPS/geranylgeranyl diphosphate synthase (GGPPS) enzyme. The apparent KM values for the substrates dimethylallyl diphosphate, geranyl diphosphate and farnesyl diphosphate were, respectively, 68 ± 5 μM, 7.8 ± 1.3 μM and 2.06 ± 0.4 μM. The protein is expressed constitutively in all intra-erythrocytic stages of P. falciparum, demonstrated by using transgenic parasites with a haemagglutinin-tagged version of FPPS. Also, the present data demonstrate that the recombinant protein is inhibited by risedronate.ConclusionsThe rPfFPPS is a bifunctional FPPS/GGPPS enzyme and the structure of products FOH and GGOH were confirmed mass spectrometry. Plasmodial FPPS represents a potential target for the rational design of chemotherapeutic agents to treat malaria.

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Section: Discussionmentioning

confidence: 99%

Cloning and characterization of bifunctional enzyme farnesyl diphosphate/geranylgeranyl diphosphate synthase from Plasmodium falciparum

Jordão

Gabriel

Alves

et al. 2013

Malar J

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“…4) (20,71). The gene encoding GGPP synthase (idsA) was cloned, identified, and expressed from Methanothermobacter marburgensis (11), Sulfolobus acidocaldarius (85,87), and Pyrococcus horikoshii (64). These enzymes are characterized by aspartate-rich motifs in their sequences, which are commonly found in polyprenyl transferases.…”

Section: Polyprenyl Diphosphate Synthesismentioning

confidence: 99%

Biosynthesis of Ether-Type Polar Lipids in Archaea and Evolutionary Considerations

Koga

Morii

2007

Microbiol Mol Biol Rev

308

263

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SUMMARY This review deals with the in vitro biosynthesis of the characteristics of polar lipids in archaea along with preceding in vivo studies. Isoprenoid chains are synthesized through the classical mevalonate pathway, as in eucarya, with minor modifications in some archaeal species. Most enzymes involved in the pathway have been identified enzymatically and/or genomically. Three of the relevant enzymes are found in enzyme families different from the known enzymes. The order of reactions in the phospholipid synthesis pathway (glycerophosphate backbone formation, linking of glycerophosphate with two radyl chains, activation by CDP, and attachment of common polar head groups) is analogous to that of bacteria. sn-Glycerol-1-phosphate dehydrogenase is responsible for the formation of the sn-glycerol-1-phosphate backbone of phospholipids in all archaea. After the formation of two ether bonds, CDP-archaeol acts as a common precursor of various archaeal phospholipid syntheses. Various phospholipid-synthesizing enzymes from archaea and bacteria belong to the same large CDP-alcohol phosphatidyltransferase family. In short, the first halves of the phospholipid synthesis pathways play a role in synthesis of the characteristic structures of archaeal and bacterial phospholipids, respectively. In the second halves of the pathways, the polar head group-attaching reactions and enzymes are homologous in both domains. These are regarded as revealing the hybrid nature of phospholipid biosynthesis. Precells proposed by Wächtershäuser are differentiated into archaea and bacteria by spontaneous segregation of enantiomeric phospholipid membranes (with sn-glycerol-1-phosphate and sn-glycerol-3-phosphate backbones) and the fusion and fission of precells. Considering the nature of the phospholipid synthesis pathways, we here propose that common phospholipid polar head groups were present in precells before the differentiation into archaea and bacteria.

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“…Plasmid pNU212 is an expression-secretion vector containing a multiple promoter region and the signal peptide-encoding region of the gene that codes for middle wall protein (MWP) (22) of B. brevis 47. pNH326 was constructed from pNH300 (16) by replacing the MWP signal peptide with a modified signal peptide, R2L6 (17), and by adding the transcription terminator of pHT926 (2). Plasmids pNU211L4PDI (8) and pMalcGG2 (15), containing the fungal PDI gene and the archaeal GGPS gene, respectively, were described previously. Plasmid pFCA-SCHL (13) contained the gene encoding the LC, a chimeric protein of the V L -C L domain of anti-11-deoxycortisol Fab and the two Fc binding domains of protein A from Staphylococcus aureus.…”

Section: Methodsmentioning

confidence: 99%

“…The amounts of the expressed proteins were routinely determined by Western blot analysis according to the method of Burnette (1), except that an anti-Fab fragment polyclonal antibody, as the primary antibody, and an alkaline phosphatase-conjugated goat anti-rabbit IgG, as the secondary antibody, were used for detection of the LC. Rabbit anti-GGPS fused to maltose binding protein antiserum (15) and an alkaline phosphataseconjugated goat anti-rabbit IgG monoclonal (FabЈ) 2 were used as the primary and secondary antibodies, respectively, for GGPS detection. BCIP-NBT (5-bromo-4-chloro-3-indolylphosphate-nitroblue tetrazolium) was used as the substrate for the color reaction.…”

Section: Methodsmentioning

confidence: 99%

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A Protein Disulfide Isomerase Gene Fusion Expression System That Increases the Extracellular Productivity of Bacillus brevis

Kajino

Ohto

Muramatsu

et al. 2000

Appl Environ Microbiol

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We have developed a versatile Bacillus brevis expression and secretion system based on the use of fungal protein disulfide isomerase (PDI) as a gene fusion partner. Fusion with PDI increased the extracellular production of heterologous proteins (light chain of immunoglobulin G, 8-fold; geranylgeranyl pyrophosphate synthase, 12-fold). Linkage to PDI prevented the aggregation of the secreted proteins, resulting in high-level accumulation of fusion proteins in soluble and biologically active forms. We also show that the disulfide isomerase activity of PDI in a fusion protein is responsible for the suppression of the aggregation of the protein with intradisulfide, whereas aggregation of the protein without intradisulfide was prevented even when the protein was fused to a mutant PDI whose two active sites were disrupted, suggesting that another PDI function, such as chaperone-like activity, synergistically prevented the aggregation of heterologous proteins in the PDI fusion expression system.A host-vector system for the efficient extracellular production of heterologous proteins, involving Bacillus brevis as the host, has been developed (22). Many proteins of bacterial origin could be produced at high levels without much difficulty. However, certain proteins, especially some mammalian ones, still exhibited low productivity. Sagiya et al. improved the B. brevis protein expression system by means such as modification of the signal sequence (17) and isolation of a protease-deficient mutant (7), which was successfully applied to the secretion and accumulation of not only prokaryotic but also eukaryotic proteins. In fact, a change of the signal peptide sequence in the B. brevis system resulted in the higher secretion of many proteins, for example, growth hormones (7, 17), interleukin 2 (21), and protein disulfide isomerase (PDI) (8), and the use of proteasedeficient mutants increased the production of extracellular proteins (6, 7). Still another way of increasing protein productivity is to link the gene of interest to a second gene which is already known to be expressed well in the host to generate a fusion protein (9). In most of the successful fusion protein systems the protein of interest is positioned at the C-terminal end of the highly expressed fusion partner (12, 19) to ensure efficient translation initiation. The thioredoxin gene fusion system also provided the solution for another major problem which has bedeviled heterologous gene expression in E. coli, i.e., the formation of inclusion bodies (11). PDI catalyzes the formation, reduction, and isomerization of disulfide bonds in vitro (5) and facilitates the folding of disulfide-bonded proteins in vivo (18). Even though the physiological roles of these multiple functions of PDI during protein folding in the cell remain obscure, PDI, promoting in vitro folding, can be used to improve an expression system for foreign genes. In fact, in a coexpression system, PDI exhibits chaperone-like activity which suppresses the aggregation and increases the yields of heterologou...

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Overexpression of an Archaeal Geranylgeranyl Diphosphate Synthase inEscherichia coliCells

Cited by 16 publications

References 11 publications

Cloning and characterization of bifunctional enzyme farnesyl diphosphate/geranylgeranyl diphosphate synthase from Plasmodium falciparum

Cloning and characterization of bifunctional enzyme farnesyl diphosphate/geranylgeranyl diphosphate synthase from Plasmodium falciparum

Biosynthesis of Ether-Type Polar Lipids in Archaea and Evolutionary Considerations

A Protein Disulfide Isomerase Gene Fusion Expression System That Increases the Extracellular Productivity of Bacillus brevis

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