Fructose 1-phosphate is the one and only physiological effector of the Cra (FruR) regulator of Pseudomonas putida
Fructose-1-phosphate (F1P) is the preferred effector of the catabolite repressor/activator (Cra) protein of the soil bacterium Pseudomonas putida but its ability to bind other metabolic intermediates in vivo is unclear. The Cra protein of this microorganism (CraPP) was submitted to mobility shift as...
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doaj-da78fb9c0fd04bf2bca8dc91931dd3712020-11-25T03:57:43ZengWileyFEBS Open Bio2211-54632014-01-014C37738610.1016/j.fob.2014.03.013Fructose 1-phosphate is the one and only physiological effector of the Cra (FruR) regulator of Pseudomonas putidaMax Chavarría0Gonzalo Durante-Rodríguez1Tino Krell2César Santiago3Jan Brezovsky4Jiri Damborsky5Víctor de Lorenzo6Systems and Synthetic Biology Program, Centro Nacional de Biotecnología (CNB-CSIC), Cantoblanco, Madrid 28049, SpainSystems and Synthetic Biology Program, Centro Nacional de Biotecnología (CNB-CSIC), Cantoblanco, Madrid 28049, SpainDepartment of Environmental Protection, Estación Experimental del Zaidín, CSIC, C/Profesor Albareda, Granada, SpainX-ray Crystallography Unit, Centro Nacional de Biotecnología (CNB-CSIC), Cantoblanco, Madrid 28049, SpainLoschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment (RECETOX), Faculty of Science, Masaryk University, Kamenice 5/A13, 625 00 Brno, Czech RepublicLoschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment (RECETOX), Faculty of Science, Masaryk University, Kamenice 5/A13, 625 00 Brno, Czech RepublicSystems and Synthetic Biology Program, Centro Nacional de Biotecnología (CNB-CSIC), Cantoblanco, Madrid 28049, SpainFructose-1-phosphate (F1P) is the preferred effector of the catabolite repressor/activator (Cra) protein of the soil bacterium Pseudomonas putida but its ability to bind other metabolic intermediates in vivo is unclear. The Cra protein of this microorganism (CraPP) was submitted to mobility shift assays with target DNA sequences (the PfruB promoter) and candidate effectors fructose-1,6-bisphosphate (FBP), glucose 6-phosphate (G6P), and fructose-6-phosphate (F6P). 1 mM F1P was sufficient to release most of the Cra protein from its operators but more than 10 mM of FBP or G6P was required to free the same complex. However, isothermal titration microcalorimetry failed to expose any specific interaction between CraPP and FBP or G6P. To solve this paradox, transcriptional activity of a PfruB-lacZ fusion was measured in wild-type and ΔfruB cells growing on substrates that change the intracellular concentrations of F1P and FBP. The data indicated that PfruB activity was stimulated by fructose but not by glucose or succinate. This suggested that CraPP represses expression in vivo of the cognate fruBKA operon in a fashion dependent just on F1P, ruling out any other physiological effector. Molecular docking and dynamic simulations of the Cra-agonist interaction indicated that both metabolites can bind the repressor, but the breach in the relative affinity of CraPP for F1P vs FBP is three orders of magnitude larger than the equivalent distance in the Escherichia coli protein. This assigns the Cra protein of P. putida the sole role of transducing the presence of fructose in the medium into a variety of direct and indirect physiological responses.http://www.sciencedirect.com/science/article/pii/S2211546314000369CraFruRPseudomonas putidaFructose 1-phosphateFructose operon |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Max Chavarría Gonzalo Durante-Rodríguez Tino Krell César Santiago Jan Brezovsky Jiri Damborsky Víctor de Lorenzo |
spellingShingle |
Max Chavarría Gonzalo Durante-Rodríguez Tino Krell César Santiago Jan Brezovsky Jiri Damborsky Víctor de Lorenzo Fructose 1-phosphate is the one and only physiological effector of the Cra (FruR) regulator of Pseudomonas putida FEBS Open Bio Cra FruR Pseudomonas putida Fructose 1-phosphate Fructose operon |
author_facet |
Max Chavarría Gonzalo Durante-Rodríguez Tino Krell César Santiago Jan Brezovsky Jiri Damborsky Víctor de Lorenzo |
author_sort |
Max Chavarría |
title |
Fructose 1-phosphate is the one and only physiological effector of the Cra (FruR) regulator of Pseudomonas putida |
title_short |
Fructose 1-phosphate is the one and only physiological effector of the Cra (FruR) regulator of Pseudomonas putida |
title_full |
Fructose 1-phosphate is the one and only physiological effector of the Cra (FruR) regulator of Pseudomonas putida |
title_fullStr |
Fructose 1-phosphate is the one and only physiological effector of the Cra (FruR) regulator of Pseudomonas putida |
title_full_unstemmed |
Fructose 1-phosphate is the one and only physiological effector of the Cra (FruR) regulator of Pseudomonas putida |
title_sort |
fructose 1-phosphate is the one and only physiological effector of the cra (frur) regulator of pseudomonas putida |
publisher |
Wiley |
series |
FEBS Open Bio |
issn |
2211-5463 |
publishDate |
2014-01-01 |
description |
Fructose-1-phosphate (F1P) is the preferred effector of the catabolite repressor/activator (Cra) protein of the soil bacterium Pseudomonas putida but its ability to bind other metabolic intermediates in vivo is unclear. The Cra protein of this microorganism (CraPP) was submitted to mobility shift assays with target DNA sequences (the PfruB promoter) and candidate effectors fructose-1,6-bisphosphate (FBP), glucose 6-phosphate (G6P), and fructose-6-phosphate (F6P). 1 mM F1P was sufficient to release most of the Cra protein from its operators but more than 10 mM of FBP or G6P was required to free the same complex. However, isothermal titration microcalorimetry failed to expose any specific interaction between CraPP and FBP or G6P. To solve this paradox, transcriptional activity of a PfruB-lacZ fusion was measured in wild-type and ΔfruB cells growing on substrates that change the intracellular concentrations of F1P and FBP. The data indicated that PfruB activity was stimulated by fructose but not by glucose or succinate. This suggested that CraPP represses expression in vivo of the cognate fruBKA operon in a fashion dependent just on F1P, ruling out any other physiological effector. Molecular docking and dynamic simulations of the Cra-agonist interaction indicated that both metabolites can bind the repressor, but the breach in the relative affinity of CraPP for F1P vs FBP is three orders of magnitude larger than the equivalent distance in the Escherichia coli protein. This assigns the Cra protein of P. putida the sole role of transducing the presence of fructose in the medium into a variety of direct and indirect physiological responses. |
topic |
Cra FruR Pseudomonas putida Fructose 1-phosphate Fructose operon |
url |
http://www.sciencedirect.com/science/article/pii/S2211546314000369 |
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