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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

RsFPPS enzyme kinetics.Purified recombinant RsFPPS was subjected to enzyme assays to determine the effect of varying concentrations of the allylic substrates A) DMAPP and C) GPP on enzyme activity. Parallel reactions using IPP as the counter substrate against B) DMAPP (50 μM) and D) GPP (100 μM) were also conducted. Inset values (A-D) are the means (±S.D.) of the derived kinetic constants Km (μM) and kcat(min−1). Values for kcat were calculated using the dimeric form of the enzyme.
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f5: RsFPPS enzyme kinetics.Purified recombinant RsFPPS was subjected to enzyme assays to determine the effect of varying concentrations of the allylic substrates A) DMAPP and C) GPP on enzyme activity. Parallel reactions using IPP as the counter substrate against B) DMAPP (50 μM) and D) GPP (100 μM) were also conducted. Inset values (A-D) are the means (±S.D.) of the derived kinetic constants Km (μM) and kcat(min−1). Values for kcat were calculated using the dimeric form of the enzyme.

Mentions: Varying concentrations of the substrates IPP, DMAPP, and GPP were used to determine the steady state kinetic constants for recombinant RsFPPS (Fig. 5A-D). For reactions where the IPP concentration was kept constant (50 μM) and DMAPP or GPP were used as counter substrates, the Km value for DMAPP was 6-fold lower than the derived Km value for GPP (Fig. 5A,C). In parallel reactions where the DMAPP (50 μM) or GPP (100 μM) concentrations were kept constant and IPP was used as a counter substrate, Km values for IPP did not differ significantly (Fig. 5B,D). It was noted though, that at higher concentrations of IPP (200 μM), decreases in enzyme activity were observed. Because of this observation, the enzyme-inhibitor constant (Ki) for substrate inhibition in these set of reactions was determined, and it revealed that reactions with DMAPP had around two-fold higher Ki values for IPP than reactions with GPP (Fig. S5). Conversely, in terms of the turnover rate (kcat) of RsFPPS, reactions where GPP was used as the counter substrate (Fig. 5C,D) showed kcat values that were 10-fold higher compared to reactions with DMAPP as the counter substrate (Fig. 5A,B), indicating that FPP is more efficiently produced when GPP is used as its allylic substrate.


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)

RsFPPS enzyme kinetics.Purified recombinant RsFPPS was subjected to enzyme assays to determine the effect of varying concentrations of the allylic substrates A) DMAPP and C) GPP on enzyme activity. Parallel reactions using IPP as the counter substrate against B) DMAPP (50 μM) and D) GPP (100 μM) were also conducted. Inset values (A-D) are the means (±S.D.) of the derived kinetic constants Km (μM) and kcat(min−1). Values for kcat were calculated using the dimeric form of the enzyme.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: RsFPPS enzyme kinetics.Purified recombinant RsFPPS was subjected to enzyme assays to determine the effect of varying concentrations of the allylic substrates A) DMAPP and C) GPP on enzyme activity. Parallel reactions using IPP as the counter substrate against B) DMAPP (50 μM) and D) GPP (100 μM) were also conducted. Inset values (A-D) are the means (±S.D.) of the derived kinetic constants Km (μM) and kcat(min−1). Values for kcat were calculated using the dimeric form of the enzyme.
Mentions: Varying concentrations of the substrates IPP, DMAPP, and GPP were used to determine the steady state kinetic constants for recombinant RsFPPS (Fig. 5A-D). For reactions where the IPP concentration was kept constant (50 μM) and DMAPP or GPP were used as counter substrates, the Km value for DMAPP was 6-fold lower than the derived Km value for GPP (Fig. 5A,C). In parallel reactions where the DMAPP (50 μM) or GPP (100 μM) concentrations were kept constant and IPP was used as a counter substrate, Km values for IPP did not differ significantly (Fig. 5B,D). It was noted though, that at higher concentrations of IPP (200 μM), decreases in enzyme activity were observed. Because of this observation, the enzyme-inhibitor constant (Ki) for substrate inhibition in these set of reactions was determined, and it revealed that reactions with DMAPP had around two-fold higher Ki values for IPP than reactions with GPP (Fig. S5). Conversely, in terms of the turnover rate (kcat) of RsFPPS, reactions where GPP was used as the counter substrate (Fig. 5C,D) showed kcat values that were 10-fold higher compared to reactions with DMAPP as the counter substrate (Fig. 5A,B), indicating that FPP is more efficiently produced when GPP is used as its allylic substrate.

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