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Cloning and characterization of farnesyl pyrophosphate synthase from the highly branched isoprenoid producing diatom Rhizosolenia setigera.

Ferriols VM, Yaginuma R, Adachi M, Takada K, Matsunaga S, Okada S - Sci Rep (2015)

Bottom Line: A reduction in HBI content from diatoms treated with an FPPS inhibitor, risedronate, suggested that RsFPPS supplies precursors for HBI biosynthesis.Furthermore, RsFPPS gene expression at various life stages of R. setigera in relation to auxosporulation were also analyzed.Herein, we present data on the possible role of RsFPPS in HBI biosynthesis, and it is to our knowledge the first instance that an FPPS was cloned and characterized from a diatom.

View Article: PubMed Central - PubMed

Affiliation: 1] Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan [2] Institute of Aquaculture, University of the Philippines Visayas, Philippines.

ABSTRACT
The diatom Rhizosolenia setigera Brightwell produces highly branched isoprenoid (HBI) hydrocarbons that are ubiquitously present in marine environments. The hydrocarbon composition of R. setigera varies between C25 and C30 HBIs depending on the life cycle stage with regard to auxosporulation. To better understand how these hydrocarbons are biosynthesized, we characterized the farnesyl pyrophosphate (FPP) synthase (FPPS) enzyme of R. setigera. An isolated 1465-bp cDNA clone contained an open reading frame spanning 1299-bp encoding a protein with 432 amino acid residues. Expression of the RsFPPS cDNA coding region in Escherichia coli produced a protein that exhibited FPPS activity in vitro. A reduction in HBI content from diatoms treated with an FPPS inhibitor, risedronate, suggested that RsFPPS supplies precursors for HBI biosynthesis. Product analysis by gas chromatography-mass spectrometry also revealed that RsFPPS produced small amounts of the cis-isomers of geranyl pyrophosphate and FPP, candidate precursors for the cis-isomers of HBIs previously characterized. Furthermore, RsFPPS gene expression at various life stages of R. setigera in relation to auxosporulation were also analyzed. Herein, we present data on the possible role of RsFPPS in HBI biosynthesis, and it is to our knowledge the first instance that an FPPS was cloned and characterized from a diatom.

No MeSH data available.


Related in: MedlinePlus

Multiple amino acid sequence alignment of R. setigera FPPS.Comparisons were made against those from mouse, yeast, bacteria, and plants. Conserved domains typical of FPP synthases are marked by Roman numerals (I-VII) and a bar above the sequences. The first and second aspartate rich regions in domains II and VI respectively are indicated by boxes.
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f3: Multiple amino acid sequence alignment of R. setigera FPPS.Comparisons were made against those from mouse, yeast, bacteria, and plants. Conserved domains typical of FPP synthases are marked by Roman numerals (I-VII) and a bar above the sequences. The first and second aspartate rich regions in domains II and VI respectively are indicated by boxes.

Mentions: The predicted molecular weight of R. setigera FPPS is 48.94 kDa, which falls within the range for those from mammals (~48 kDa)2728, and is relatively larger than those from plants and fungi (39-44 kDa)20212229, and bacteria (~32 kDa)30. A BLAST search against the NCBI online protein database showed that the deduced RsFPPS amino acid sequence shared 58% and 55% identity with annotated FPPSs from eustigmatophytes and brown algae, respectively, and 46-51% identity with representative FPP synthase sequences from other organisms (animals, higher plants, green algae, and fungi). Phylogenetic analysis of RsFPPS showed that it was more closely related to algal FPP synthase than to those of animals, higher plants, fungi, or bacteria based on sequence conservation (Fig. 2). Sequence alignment of the putative amino acid sequence of RsFPPS against those of mammals, plants, yeast, and bacteria (Fig. 3) revealed that it contained all conserved amino acid residues necessary for substrate binding and catalytic activity (domains I-VII) typical of other identified FPPSs31. Two characteristic aspartate-rich motifs were also present in the RsFPPS sequence (Fig. 3).


Cloning and characterization of farnesyl pyrophosphate synthase from the highly branched isoprenoid producing diatom Rhizosolenia setigera.

Ferriols VM, Yaginuma R, Adachi M, Takada K, Matsunaga S, Okada S - Sci Rep (2015)

Multiple amino acid sequence alignment of R. setigera FPPS.Comparisons were made against those from mouse, yeast, bacteria, and plants. Conserved domains typical of FPP synthases are marked by Roman numerals (I-VII) and a bar above the sequences. The first and second aspartate rich regions in domains II and VI respectively are indicated by boxes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4440519&req=5

f3: Multiple amino acid sequence alignment of R. setigera FPPS.Comparisons were made against those from mouse, yeast, bacteria, and plants. Conserved domains typical of FPP synthases are marked by Roman numerals (I-VII) and a bar above the sequences. The first and second aspartate rich regions in domains II and VI respectively are indicated by boxes.
Mentions: The predicted molecular weight of R. setigera FPPS is 48.94 kDa, which falls within the range for those from mammals (~48 kDa)2728, and is relatively larger than those from plants and fungi (39-44 kDa)20212229, and bacteria (~32 kDa)30. A BLAST search against the NCBI online protein database showed that the deduced RsFPPS amino acid sequence shared 58% and 55% identity with annotated FPPSs from eustigmatophytes and brown algae, respectively, and 46-51% identity with representative FPP synthase sequences from other organisms (animals, higher plants, green algae, and fungi). Phylogenetic analysis of RsFPPS showed that it was more closely related to algal FPP synthase than to those of animals, higher plants, fungi, or bacteria based on sequence conservation (Fig. 2). Sequence alignment of the putative amino acid sequence of RsFPPS against those of mammals, plants, yeast, and bacteria (Fig. 3) revealed that it contained all conserved amino acid residues necessary for substrate binding and catalytic activity (domains I-VII) typical of other identified FPPSs31. Two characteristic aspartate-rich motifs were also present in the RsFPPS sequence (Fig. 3).

Bottom Line: A reduction in HBI content from diatoms treated with an FPPS inhibitor, risedronate, suggested that RsFPPS supplies precursors for HBI biosynthesis.Furthermore, RsFPPS gene expression at various life stages of R. setigera in relation to auxosporulation were also analyzed.Herein, we present data on the possible role of RsFPPS in HBI biosynthesis, and it is to our knowledge the first instance that an FPPS was cloned and characterized from a diatom.

View Article: PubMed Central - PubMed

Affiliation: 1] Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan [2] Institute of Aquaculture, University of the Philippines Visayas, Philippines.

ABSTRACT
The diatom Rhizosolenia setigera Brightwell produces highly branched isoprenoid (HBI) hydrocarbons that are ubiquitously present in marine environments. The hydrocarbon composition of R. setigera varies between C25 and C30 HBIs depending on the life cycle stage with regard to auxosporulation. To better understand how these hydrocarbons are biosynthesized, we characterized the farnesyl pyrophosphate (FPP) synthase (FPPS) enzyme of R. setigera. An isolated 1465-bp cDNA clone contained an open reading frame spanning 1299-bp encoding a protein with 432 amino acid residues. Expression of the RsFPPS cDNA coding region in Escherichia coli produced a protein that exhibited FPPS activity in vitro. A reduction in HBI content from diatoms treated with an FPPS inhibitor, risedronate, suggested that RsFPPS supplies precursors for HBI biosynthesis. Product analysis by gas chromatography-mass spectrometry also revealed that RsFPPS produced small amounts of the cis-isomers of geranyl pyrophosphate and FPP, candidate precursors for the cis-isomers of HBIs previously characterized. Furthermore, RsFPPS gene expression at various life stages of R. setigera in relation to auxosporulation were also analyzed. Herein, we present data on the possible role of RsFPPS in HBI biosynthesis, and it is to our knowledge the first instance that an FPPS was cloned and characterized from a diatom.

No MeSH data available.


Related in: MedlinePlus