Increasing the intracellular isoprenoid pool in Saccharomyces cerevisiae by structural fine-tuning of a bifunctional farnesyl diphosphate synthase

Sebastián Rubat, Ignacio Varas, Romina Sepúlveda, Daniel Almonacid, Fernando González-Nilo, Eduardo Agosin

Resultado de la investigación: Article

1 Cita (Scopus)

Resumen

Farnesyl diphosphate synthase (FPPS) is a key enzyme responsible for the supply of isoprenoid precursors for several essential metabolites, including sterols, dolichols and ubiquinone. In Saccharomyces cerevisiae, FPPS catalyzes the sequential condensation of two molecules of isopentenyl diphosphate (IPP) with dimethylallyl diphosphate (DMAPP), producing geranyl diphosphate (GPP) and farnesyl diphosphate (FPP). Critical amino acid residues that determine product chain length were determined by a comparative study of strict GPP synthases versus strict FPPS. In silico ΔΔG, i.e. differential binding energy between a protein and two different ligands-of yeast FPPS mutants was evaluated, and F96, A99 and E165 residues were identified as key determinants for product selectivity. A99X variants were evaluated in vivo, S. cerevisiae strains carrying A99R and A99H variants showed significant differences on GPP concentrations and specific growth rates. The FPPS A99T variant produced unquantifiable amounts of FPP and no effect on GPP production was observed. Strains carrying A99Q, A99Y and A99K FPPS accumulated high amounts of DMAPP-IPP, with a decrease in GPP and FPP. Our results demonstrated the relevance of the first residue before FARM (First Aspartate Rich Motif) over substrate consumption and product specificity of S. cerevisiae FPPS in vivo. The presence of A99H significantly modified product selectivity and appeared to be relevant for GPP synthesis.

Idioma originalEnglish
PublicaciónFEMS Yeast Research
Volumen17
N.º4
DOI
EstadoPublished - 1 jun 2017

Huella dactilar

Geranyltranstransferase
Terpenes
Saccharomyces cerevisiae
Dolichol
Ubiquinone
Sterols
Aspartic Acid
Computer Simulation
Yeasts
Ligands
Amino Acids

ASJC Scopus subject areas

  • Microbiology
  • Applied Microbiology and Biotechnology

Citar esto

Rubat, Sebastián ; Varas, Ignacio ; Sepúlveda, Romina ; Almonacid, Daniel ; González-Nilo, Fernando ; Agosin, Eduardo. / Increasing the intracellular isoprenoid pool in Saccharomyces cerevisiae by structural fine-tuning of a bifunctional farnesyl diphosphate synthase. En: FEMS Yeast Research. 2017 ; Vol. 17, N.º 4.
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Increasing the intracellular isoprenoid pool in Saccharomyces cerevisiae by structural fine-tuning of a bifunctional farnesyl diphosphate synthase. / Rubat, Sebastián; Varas, Ignacio; Sepúlveda, Romina; Almonacid, Daniel; González-Nilo, Fernando; Agosin, Eduardo.

En: FEMS Yeast Research, Vol. 17, N.º 4, 01.06.2017.

Resultado de la investigación: Article

TY - JOUR

T1 - Increasing the intracellular isoprenoid pool in Saccharomyces cerevisiae by structural fine-tuning of a bifunctional farnesyl diphosphate synthase

AU - Rubat, Sebastián

AU - Varas, Ignacio

AU - Sepúlveda, Romina

AU - Almonacid, Daniel

AU - González-Nilo, Fernando

AU - Agosin, Eduardo

PY - 2017/6/1

Y1 - 2017/6/1

N2 - Farnesyl diphosphate synthase (FPPS) is a key enzyme responsible for the supply of isoprenoid precursors for several essential metabolites, including sterols, dolichols and ubiquinone. In Saccharomyces cerevisiae, FPPS catalyzes the sequential condensation of two molecules of isopentenyl diphosphate (IPP) with dimethylallyl diphosphate (DMAPP), producing geranyl diphosphate (GPP) and farnesyl diphosphate (FPP). Critical amino acid residues that determine product chain length were determined by a comparative study of strict GPP synthases versus strict FPPS. In silico ΔΔG, i.e. differential binding energy between a protein and two different ligands-of yeast FPPS mutants was evaluated, and F96, A99 and E165 residues were identified as key determinants for product selectivity. A99X variants were evaluated in vivo, S. cerevisiae strains carrying A99R and A99H variants showed significant differences on GPP concentrations and specific growth rates. The FPPS A99T variant produced unquantifiable amounts of FPP and no effect on GPP production was observed. Strains carrying A99Q, A99Y and A99K FPPS accumulated high amounts of DMAPP-IPP, with a decrease in GPP and FPP. Our results demonstrated the relevance of the first residue before FARM (First Aspartate Rich Motif) over substrate consumption and product specificity of S. cerevisiae FPPS in vivo. The presence of A99H significantly modified product selectivity and appeared to be relevant for GPP synthesis.

AB - Farnesyl diphosphate synthase (FPPS) is a key enzyme responsible for the supply of isoprenoid precursors for several essential metabolites, including sterols, dolichols and ubiquinone. In Saccharomyces cerevisiae, FPPS catalyzes the sequential condensation of two molecules of isopentenyl diphosphate (IPP) with dimethylallyl diphosphate (DMAPP), producing geranyl diphosphate (GPP) and farnesyl diphosphate (FPP). Critical amino acid residues that determine product chain length were determined by a comparative study of strict GPP synthases versus strict FPPS. In silico ΔΔG, i.e. differential binding energy between a protein and two different ligands-of yeast FPPS mutants was evaluated, and F96, A99 and E165 residues were identified as key determinants for product selectivity. A99X variants were evaluated in vivo, S. cerevisiae strains carrying A99R and A99H variants showed significant differences on GPP concentrations and specific growth rates. The FPPS A99T variant produced unquantifiable amounts of FPP and no effect on GPP production was observed. Strains carrying A99Q, A99Y and A99K FPPS accumulated high amounts of DMAPP-IPP, with a decrease in GPP and FPP. Our results demonstrated the relevance of the first residue before FARM (First Aspartate Rich Motif) over substrate consumption and product specificity of S. cerevisiae FPPS in vivo. The presence of A99H significantly modified product selectivity and appeared to be relevant for GPP synthesis.

KW - FPPS

KW - isoprenoid

KW - LC-MS

KW - protein engineering

KW - single-point mutation

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U2 - 10.1093/femsyr/fox032

DO - 10.1093/femsyr/fox032

M3 - Article

C2 - 28854674

AN - SCOPUS:85046986546

VL - 17

JO - FEMS Yeast Research

JF - FEMS Yeast Research

SN - 1567-1356

IS - 4

ER -