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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

GC/MS total ion chromatogram of enzyme reaction products using IPP and DMAPP as substrates.Upper panels show prominent peaks corresponding to A) geraniol (GOH) and B) E,E-farnesol (E,E-FOH) with minor peaks for nerol (NOH) and Z,E-farnesol (Z,E-FOH) marked with asterisks (*). Lower panels are magnified images of the boxed areas in the upper panels. Other minor peaks in the upper panels are either contaminants or artefacts of treatment with alkaline phosphatase as determined in control alkaline phosphatase reactions using GPP or E,E-FPP standards.
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f6: GC/MS total ion chromatogram of enzyme reaction products using IPP and DMAPP as substrates.Upper panels show prominent peaks corresponding to A) geraniol (GOH) and B) E,E-farnesol (E,E-FOH) with minor peaks for nerol (NOH) and Z,E-farnesol (Z,E-FOH) marked with asterisks (*). Lower panels are magnified images of the boxed areas in the upper panels. Other minor peaks in the upper panels are either contaminants or artefacts of treatment with alkaline phosphatase as determined in control alkaline phosphatase reactions using GPP or E,E-FPP standards.

Mentions: Products of large scale enzyme assays using IPP and DMAPP as substrates were further verified by GC/MS after converting the generated prenyl pyrophosphates to their corresponding alcohols using alkaline phosphatase. This was done in order to check for possible formation of reaction product cis-isomers since separation of GPP and FPP cis- and trans-isomers was difficult using the described LC/MS conditions. The formation of geraniol (GOH) and E,E-farnesol (E,E-FOH) was easily detected by GC/MS with peaks appearing at retention times of 12.45 and 18.45 min, respectively (Fig. 6A,B, upper panels; Fig. S4). Interestingly, small amounts of their cis- isomers, nerol (NOH) and Z,E-farnesol (Z,E-FOH), were also detected with retention times of 12.12 and 18.22 min, respectively (Fig. 6A,B, lower panels; Fig. S4). When IPP and GPP were used as substrates, only a small amount of Z,E-FOH was detected and no peak corresponding to NOH was observed. The peaks for the cis-isomers were not detected when control reactions using GPP and E,E-FPP standards were treated with alkaline phosphatase indicating that the expressed RsFPPS produced a small amount of neryl pyrophosphate (NPP) and Z,E-FPP as minor products.


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)

GC/MS total ion chromatogram of enzyme reaction products using IPP and DMAPP as substrates.Upper panels show prominent peaks corresponding to A) geraniol (GOH) and B) E,E-farnesol (E,E-FOH) with minor peaks for nerol (NOH) and Z,E-farnesol (Z,E-FOH) marked with asterisks (*). Lower panels are magnified images of the boxed areas in the upper panels. Other minor peaks in the upper panels are either contaminants or artefacts of treatment with alkaline phosphatase as determined in control alkaline phosphatase reactions using GPP or E,E-FPP standards.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: GC/MS total ion chromatogram of enzyme reaction products using IPP and DMAPP as substrates.Upper panels show prominent peaks corresponding to A) geraniol (GOH) and B) E,E-farnesol (E,E-FOH) with minor peaks for nerol (NOH) and Z,E-farnesol (Z,E-FOH) marked with asterisks (*). Lower panels are magnified images of the boxed areas in the upper panels. Other minor peaks in the upper panels are either contaminants or artefacts of treatment with alkaline phosphatase as determined in control alkaline phosphatase reactions using GPP or E,E-FPP standards.
Mentions: Products of large scale enzyme assays using IPP and DMAPP as substrates were further verified by GC/MS after converting the generated prenyl pyrophosphates to their corresponding alcohols using alkaline phosphatase. This was done in order to check for possible formation of reaction product cis-isomers since separation of GPP and FPP cis- and trans-isomers was difficult using the described LC/MS conditions. The formation of geraniol (GOH) and E,E-farnesol (E,E-FOH) was easily detected by GC/MS with peaks appearing at retention times of 12.45 and 18.45 min, respectively (Fig. 6A,B, upper panels; Fig. S4). Interestingly, small amounts of their cis- isomers, nerol (NOH) and Z,E-farnesol (Z,E-FOH), were also detected with retention times of 12.12 and 18.22 min, respectively (Fig. 6A,B, lower panels; Fig. S4). When IPP and GPP were used as substrates, only a small amount of Z,E-FOH was detected and no peak corresponding to NOH was observed. The peaks for the cis-isomers were not detected when control reactions using GPP and E,E-FPP standards were treated with alkaline phosphatase indicating that the expressed RsFPPS produced a small amount of neryl pyrophosphate (NPP) and Z,E-FPP as minor products.

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