Open AccessFructose 1‐phosphate is the one and only physiological effector of the Cra (FruR) regulator of Pseudomonas putida
Author(s) -
Chavarría Max,
Durante-Rodríguez Gonzalo,
Krell Tino,
Santiago César,
Brezovsky Jan,
Damborsky Jiri,
de Lorenzo Víctor
Publication year - 2014
Publication title -
febs open bio
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.718
H-Index - 31
ISSN - 2211-5463
DOI - 10.1016/j.fob.2014.03.013
Subject(s) - pseudomonas putida , catabolite repression , repressor , effector , biochemistry , fructose , operon , glucose 6 phosphate , biology , tetr , lac operon , escherichia coli , allosteric regulation , chemistry , mutant , gene expression , gene , receptor , enzyme
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 (Cra PP ) was submitted to mobility shift assays with target DNA sequences (the P fruB 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 Cra PP and FBP or G6P. To solve this paradox, transcriptional activity of a P fruB ‐lacZ fusion was measured in wild‐type and Δ fruB cells growing on substrates that change the intracellular concentrations of F1P and FBP. The data indicated that P fruB activity was stimulated by fructose but not by glucose or succinate. This suggested that Cra PP 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 Cra PP 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.