Theresa Kouril

Integrated systems biology approach including transcriptome, metabolome, proteome analyses and modelling to elucidate amino acid degradation in S. solfataricus P2.

The gluconeogenic conversion of 3-phosphoglycerate via 1,3-bisphosphoglycerate to glyceraldehyde-3-phosphate was compared at 30 C and at 70 C. At 30 C it was possible to produce 1,3-bisphosphoglycerate from 3-phosphoglycerate with phosphoglycerate kinase, but at 70 C, 1,3- bisphosphoglycerate was dephosphorylated rapidly to 3-phosphoglycerate, effectively turning the phosphoglycerate kinase into a futile cycle. At both temperatures it was possible to convert 3-phosphoglycerate to glyceraldehyde
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Integrated systems biology approach including transcriptome, metabolome, biochemistry, proteome analyses and modelling to elucidate the catabolic pathway for L-fucose in S. solfataricus P2.

To investigate amino acid degradation pathways in Sulfolobus solfataricus transcriptome, proteome and metabolome analyses were performed on cells grown on caseinhydrolysate as carbon source. Cells grown with glucose served as reference condition. Metabolic modelling was used to compare the efficiency of different degradation routes.

PGK-GAPDH reactions were studied in vitro at 30 and 70 using yeast or Sso enzymes

SED-ML:
https://jjj.bio.vu.nl/models/experiments/kouril2017_fig4b/simulate
https://jjj.bio.vu.nl/models/experiments/kouril2017_fig4c/simulate

PGK reaction at 30 C. Yeast PGK was incubated at 30 C, in the presence or absence of the ATP recycling system, and the conversion of 3 PG to BPG was followed.
SED-ML simulations
Fig. 1A: https://jjj.bio.vu.nl/models/experiments/kouril2017_fig1a/simulate

PGK reaction at 70C. Sulfolobus solfataricus PGK was incubated at 70C in presence and absence of an ATP recycling system.
Changes in metabolite concentrations was followed via 31P NMR or enzymatic analyses.

SED-ML:
https://jjj.bio.vu.nl/models/experiments/kouril2017_fig3b/simulate
https://jjj.bio.vu.nl/models/experiments/kouril2017_fig3c/simulate
https://jjj.bio.vu.nl/models/experiments/kouril2017_fig3d/simulate

BPG produced with yeast PGK was incubated at 70 C,upon which BPG rapidly dephosphorylates to 3PG.
SED-ML:
https://jjj.bio.vu.nl/models/experiments/kouril2017_fig2b/simulate

Experimental data for the yeast PGK incubations at 30C, with and without recycling of ATP.

Changes in metabolite concentrations were either quantified via 31P NMR or enzymatically

BPG degradation at 70C

Simulation results of TPI experimental data for GAP and DHAP saturation.

Simulation results of temperature degradation of gluconeogenic intermediates

Simulation results of experimental data of the reconstituted gluconeogenic system

Simulation results of GAPDH experimental data for BPG, NADPH, NADP, GAP, and Pi

incubations with yeast enzymes at 30C

metabolite quantification via enzymatic analysis

PGK yeast without recycling of ATP

RNAseq analysis of S. solfataricus grown on either L-fucose or D-glucose

Activity assays in cell free extracts of S. solfataricus grown on either L-fucose or D-glucose and activity assays with recombinant proteins

Experimental data for 3PG conversion to fructose-6-phosphase in reconstituted systems of gluconeogenesis of S. solfataricus

Kinetic characterisation of FBPAase. Expermental data for enzyme reaction rates with increasing concentrations of DHAP and GAP.

Kinetic characterisation of TPI. Experimental data for enzyme reaction rates with increasing concentrations of DHAP and GAP, and inhibition with 3PG and PEP.

Protein extraction, iTRAQ labeling, peptides separation, mass spectrometry and data analysis

Preparation of cell free extracts of the recombinant E. coli strains expressing the gluconeogenic S. solfataricus enzymes.

Cloning and heterologous expression of gluconeogenic enzymes from S. solfataricus in E. coli

Purification of gluconeogenic enzymes from S. solfataricus in recombinant E.coli extracts

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