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- Intermediate instability at high temperature leads to low pathway efficiency for an in vitro reconstituted system of gluconeogenesis in Sulfolobus solfataricus

**Abstract:**

Four enzymes of the gluconeogenic pathway in Sulfolobus solfataricus were purified and kinetically characterized. The enzymes were reconstituted in vitro to quantify the contribution of temperature instability of the pathway intermediates to carbon loss from the system. The reconstituted system, consisting of phosphoglycerate kinase, glyceraldehyde 3-phosphate dehydrogenase, triose phosphate isomerase and the fructose 1,6-bisphosphate aldolase/ phosphatase maintained a constant consumption rate of 3-phosphoglycerate and production of fructose 6-phosphate over a 1 hour period. Cofactors ATP and NADPH were regenerated via pyruvate kinase and glucose dehydrogenase. A mathematical model was constructed on the basis of the kinetics of the purified enzymes and the measured half-life times of the pathway intermediates. The model quantitatively predicted the systems uxes and metabolite concentrations. Relative enzyme concentrations were chosen such that half the carbon in the system was lost due to degradation of the thermolabile intermediates dihydroxyacetone phosphate, glyceraldehyde 3-phosphate and 1,3 bisphosphoglycerate, indicating that intermediate instability at high temperature can significantly affect pathway efficiency. This article is protected by copyright. All rights reserved.

**SEEK ID:** https://fairdomhub.org/publications/213

**PubMed ID:**
23865479

**Projects:** SulfoSys

**Journal:**
FEBS J.

**Citation:**

**Date Published:**
20th Jul 2013

**Authors:**
Theresa Kouril, Dominik Esser, Julia Kort, Hans Westerhoff, Bettina Siebers, Jacky Snoep

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**Projects:** SulfoSys, SulfoSys - Biotec, HOTSOLUTE, Computational pathway design for biotechnological applications, ScyCode

**Institutions:** University Duisburg-Essen, Universitity Duisburg-Essen

**Roles:** Project Coordinator, Vice Coordinator

**Expertise:** Archaeal physiology, Hyperthermophiles, Thermoacidohiles, Biotechnology, Carbohydrate Metabolism of Archaea, Reconstruction of metabolic pathways, Regulation of carbohydrate metabolism

**Tools:** Microbiology, Physiology, Systems Biology, Molecular Biology, Biochemistry

Head of the group of Molecular Enzyme Technology and Biochemistry (Faculty of Chemistry) at the University of Duisburg-Essen. My research interest is on archaeal physiology with a special focuss on the central carbohydrate metabolism of (hyper)thermophilic Archaea and its regulation. The aim is to gain a systems level understanding by the combination of modern highthrouput analyses with classical biochemistry and molecular biology.

Archaea possess many novel enzymes and pathways and our aim is

...

**Projects:** SulfoSys, SulfoSys - Biotec

**Institutions:** University Duisburg-Essen

**Projects:** PSYSMO, MOSES, SysMO DB, SysMO-LAB, SulfoSys, SulfoSys - Biotec, Whole body modelling of glucose metabolism in malaria patients, FAIRDOM, Molecular Systems Biology, COMBINE Multicellular Modelling, HOTSOLUTE, Steroid biosynthesis, Yeast glycolytic oscillations, Computational pathway design for biotechnological applications

**Institutions:** Manchester Centre for Integrative Systems Biology, University of Manchester, University of Stellenbosch, School of Computer Science, University of Manchester, Stellenbosch University

Silicon cell model for the central carbohydrate metabolism of the archaeon Sulfolobus solfataricus under temperature variation

**Programme**: SysMO

**Public web page**: http://sulfosys.com/

**Organisms:** Sulfolobus solfataricus

Temperature degradation of BPG, GAP and DHAP

**Contributor**: Jacky Snoep

**Assay type**: Metabolite Concentration

**Technology type**: Technology Type

**Snapshots: **No snapshots

**Investigation:** Central Carbon Metabolism of Sulfolobus solfata...

**Study:** Model Gluconeogenesis

**Organisms**: Sulfolobus solfataricus

**SOPs:** No SOPs

**Data files:** Temperature Degradation of Gluconeogenic Interm...

Kinetic characterisation of triose phosphate isomerase

**Contributor**: Jacky Snoep

**Assay type**: Enzymatic Assay

**Technology type**: Initial Rate Experiment

**Snapshots: **No snapshots

**Investigation:** Central Carbon Metabolism of Sulfolobus solfata...

**Study:** Model Gluconeogenesis

**Organisms**: Sulfolobus solfataricus

**SOPs:** Cloning and heterologous expression in E. coli, Preparation of cell extracts of recombinant E.coli, Protein purification from E. coli extracts

**Data files:** TPI Kinetic Data

Kinetic characterisation of glyceraldehyde 3-phosphate dehydrogenase

**Contributor**: Jacky Snoep

**Assay type**: Enzymatic Assay

**Technology type**: Initial Rate Experiment

**Snapshots: **No snapshots

**Investigation:** Central Carbon Metabolism of Sulfolobus solfata...

**Study:** Model Gluconeogenesis

**Organisms**: Sulfolobus solfataricus

**SOPs:** Cloning and heterologous expression in E. coli, Preparation of cell extracts of recombinant E.coli, Protein purification from E. coli extracts

**Data files:** GAPDH Kinetic Data

Kinetic characterisation of fructose 1,6-bisphosphate aldolase phosphatase

**Contributor**: Jacky Snoep

**Assay type**: Enzymatic Assay

**Technology type**: Initial Rate Experiment

**Snapshots: **No snapshots

**Investigation:** Central Carbon Metabolism of Sulfolobus solfata...

**Study:** Model Gluconeogenesis

**Organisms**: Sulfolobus solfataricus

**SOPs:** Cloning and heterologous expression in E. coli, Preparation of cell extracts of recombinant E.coli, Protein purification from E. coli extracts

**Data files:** FBPAase Kinetic data

Kinetic characterisation of phosphoglycerate kinase

**Contributor**: Jacky Snoep

**Assay type**: Enzymatic Assay

**Technology type**: Initial Rate Experiment

**Snapshots: **No snapshots

**Investigation:** Central Carbon Metabolism of Sulfolobus solfata...

**Study:** Model Gluconeogenesis

**Organisms**: Sulfolobus solfataricus

**SOPs:** Cloning and heterologous expression in E. coli, Preparation of cell extracts of recombinant E.coli, Protein purification from E. coli extracts

**Data files:** PGK Kinetic Data

Mathematical model for PGK kinetics, saturation with ADP, ATP, 3PG and BPG.

**Investigation:** Central Carbon Metabolism of Sulfolobus solfata...

**Study:** Model Gluconeogenesis

**Organisms**: Sulfolobus solfataricus

**Models:** PGK Kinetic Model

**SOPs:** No SOPs

**Data files:** PGK Kinetic Data, PGK Kinetics Simulation

Mathematical model for TPI kinetics, saturation with GAP and DHAP, and inhibition by 3PG and PEP

**Investigation:** Central Carbon Metabolism of Sulfolobus solfata...

**Study:** Model Gluconeogenesis

**Organisms**: Sulfolobus solfataricus

**Models:** TPI Kinetic Model

**SOPs:** No SOPs

**Data files:** TPI Kinetic Data, TPI Kinetics Simulation

Modelling the degradation of BPG, GAP and DHAP at high temperature

**Investigation:** Central Carbon Metabolism of Sulfolobus solfata...

**Study:** Model Gluconeogenesis

**Organisms**: Sulfolobus solfataricus

**Models:** Kinetic Model of Temperature Degradation of Glu...

**SOPs:** No SOPs

**Data files:** Temperature Degradation Kinetics Simulation, Temperature Degradation of Gluconeogenic Interm...

Mathematical model for FBPAase kinetics, saturation with DHAP and GAP

**Investigation:** Central Carbon Metabolism of Sulfolobus solfata...

**Study:** Model Gluconeogenesis

**Organisms**: Sulfolobus solfataricus

**Models:** FBPAase Kinetic Model

**SOPs:** No SOPs

**Data files:** FBPAase Kinetic data, FBPAase Kinetics Simulation

Mathematical model for GAPDH kinetics, saturation with BPG, NADPH, NADP, GAP and Pi

**Investigation:** Central Carbon Metabolism of Sulfolobus solfata...

**Study:** Model Gluconeogenesis

**Organisms**: Sulfolobus solfataricus

**Models:** GAPDH Kinetic Model

**SOPs:** No SOPs

**Data files:** GAPDH Kinetic Data, GAPDH Kinetics Simulation

In vitro reconstitution of the PGK, GAPHD, TPI and FBPAase enzymes from S. solfataricus

**Investigation:** Central Carbon Metabolism of Sulfolobus solfata...

**Study:** Model Gluconeogenesis

**Organisms**: Sulfolobus solfataricus

**Models:** FBPAase Kinetic Model, GAPDH Kinetic Model, Kinetic Model of Temperature Degradation of Glu..., Model of reconstituted gluconeogenesis system i..., PGK Kinetic Model, TPI Kinetic Model

**SOPs:** No SOPs

**Data files:** No Data files

Model prediction of the conversion of 3PG to fructose-6-phosphate and the gluconeogenic pathway intermediates.

https://jjj.bio.vu.nl/models/experiments/kouril3_experiment-user/simulate

**Investigation:** Central Carbon Metabolism of Sulfolobus solfata...

**Study:** Carbon Loss at High Temperature

**Organisms**: Sulfolobus solfataricus

**Models:** Model of reconstituted gluconeogenesis system i...

**SOPs:** No SOPs

**Data files:** Experimental Data for Reconstituted Gluconeogen..., Simulation for Reconstituted Gluconeogenic System

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

**Creators: **Jacky Snoep, Theresa Kouril

**Contributor**: Jacky Snoep

**Investigations:** Central Carbon Metabolism of Sulfolobus solfata...

**Studies:** Model Gluconeogenesis

**Assays:** TPI Modelling

Simulation results of temperature degradation of gluconeogenic intermediates

**Creators: **Jacky Snoep, Theresa Kouril

**Contributor**: Jacky Snoep

**Investigations:** Central Carbon Metabolism of Sulfolobus solfata...

**Studies:** Model Gluconeogenesis

Simulation results of experimental data of the reconstituted gluconeogenic system

**Creators: **Jacky Snoep, Theresa Kouril

**Contributor**: Jacky Snoep

Simulation results of PGK experimental data for ADP, ATP, 3PG and BPG saturation.

**Creator: **Jacky Snoep

**Contributor**: Jacky Snoep

**Investigations:** Central Carbon Metabolism of Sulfolobus solfata...

**Studies:** Model Gluconeogenesis

**Assays:** PGK Modelling

Simulation results of FBPAase of experimental data for DHAP and GAP saturation

**Creator: **Jacky Snoep

**Contributor**: Jacky Snoep

**Investigations:** Central Carbon Metabolism of Sulfolobus solfata...

**Studies:** Model Gluconeogenesis

**Assays:** FBPAase Modelling

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

**Creators: **Jacky Snoep, Theresa Kouril

**Contributor**: Jacky Snoep

**Investigations:** Central Carbon Metabolism of Sulfolobus solfata...

**Studies:** Model Gluconeogenesis

**Assays:** GAPDH Modelling

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

**Creators: **Jacky Snoep, Theresa Kouril

**Contributor**: Jacky Snoep

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

**Creators: **Jacky Snoep, Theresa Kouril

**Contributor**: Jacky Snoep

**Investigations:** Central Carbon Metabolism of Sulfolobus solfata...

**Studies:** Model Gluconeogenesis

**Assays:** FBPAase, FBPAase Modelling

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

**Creators: **Jacky Snoep, Theresa Kouril

**Contributor**: Jacky Snoep

**Investigations:** Central Carbon Metabolism of Sulfolobus solfata...

**Studies:** Model Gluconeogenesis

**Assays:** TPI, TPI Modelling

Kinetic characterisation of GAPDH. Expermental data for enzyme reaction rates with increasing concentrations of BPG, NADPH, NADP, GAP and Pi.

**Creators: **Jacky Snoep, Theresa Kouril

**Contributor**: Jacky Snoep

**Investigations:** Central Carbon Metabolism of Sulfolobus solfata...

**Studies:** Model Gluconeogenesis

**Assays:** GAPDH, GAPDH Modelling

Experimental data for determination of half-life of gluconeogenic intermediates (BPG, GAP and DHAP).

**Creators: **Jacky Snoep, Theresa Kouril

**Contributor**: Jacky Snoep

Kinetic characterisation of PGK. Expermental data for enzyme reaction rates with increasing concentrations of ATP, ADP, BPG and 3PG.

**Creators: **Jacky Snoep, Theresa Kouril

**Contributor**: Jacky Snoep

**Investigations:** Central Carbon Metabolism of Sulfolobus solfata...

**Studies:** Model Gluconeogenesis

**Assays:** PGK, PGK Modelling

Model of reconstituted gluconeogenesis system in S. solfataricus based on the individual kinetic models for PGK, GAPDH, TPI, FBPAase.

**Creator: **Jacky Snoep

**Contributor**: Jacky Snoep

**Model type**: Ordinary differential equations (ODE)

**Model format**: SBML

**Environment**: JWS Online

**Organism**: Sulfolobus solfataricus

**Investigations:** Central Carbon Metabolism of Sulfolobus solfata...

**Studies:** Carbon Loss at High Temperature, Model Gluconeogenesis

**Modelling analyses:** Model Validation Gluconeogenesis in S. solfatar..., Reconstituted Gluconeogenesis System

Exponential decay model of gluconeogenic intermediates

**Creator: **Jacky Snoep

**Contributor**: Jacky Snoep

**Model type**: Ordinary differential equations (ODE)

**Model format**: Mathematica

**Environment**: Not specified

**Organism**: Sulfolobus solfataricus

**Investigations:** Central Carbon Metabolism of Sulfolobus solfata...

**Studies:** Model Gluconeogenesis

**Modelling analyses:** Modelling Metabolite Degradation at High Temper..., Reconstituted Gluconeogenesis System

Mathematical model for TPI kinetics, GAP and DHAP saturation, and inhibition with 3PG and PEP.

**Creator: **Jacky Snoep

**Contributor**: Jacky Snoep

**Model type**: Ordinary differential equations (ODE)

**Model format**: Mathematica

**Environment**: Not specified

**Organism**: Sulfolobus solfataricus

**Investigations:** Central Carbon Metabolism of Sulfolobus solfata...

**Studies:** Model Gluconeogenesis

**Modelling analyses:** Reconstituted Gluconeogenesis System, TPI Modelling

Mathematical model for GAPDH kinetics, BPG, NADPH, NADP, GAP and Pi saturation.

**Creator: **Jacky Snoep

**Contributor**: Jacky Snoep

**Model type**: Ordinary differential equations (ODE)

**Model format**: Mathematica

**Environment**: Not specified

**Organism**: Sulfolobus solfataricus

**Investigations:** Central Carbon Metabolism of Sulfolobus solfata...

**Studies:** Model Gluconeogenesis

**Modelling analyses:** GAPDH Modelling, Reconstituted Gluconeogenesis System

Mathematical model for FBPAase kinetics, saturation with DHAP and GAP

**Creator: **Jacky Snoep

**Contributor**: Jacky Snoep

**Model type**: Ordinary differential equations (ODE)

**Model format**: Mathematica

**Environment**: Not specified

**Organism**: Sulfolobus solfataricus

**Investigations:** Central Carbon Metabolism of Sulfolobus solfata...

**Studies:** Model Gluconeogenesis

**Modelling analyses:** FBPAase Modelling, Reconstituted Gluconeogenesis System

Mathematical model for PGK kinetics, ADP, ATP, 3PG and BPG saturation.

**Creator: **Jacky Snoep

**Contributor**: Jacky Snoep

**Model type**: Ordinary differential equations (ODE)

**Model format**: Mathematica

**Environment**: Not specified

**Organism**: Sulfolobus solfataricus

**Investigations:** Central Carbon Metabolism of Sulfolobus solfata...

**Studies:** Model Gluconeogenesis

**Modelling analyses:** PGK Modelling, Reconstituted Gluconeogenesis System