RESUMO
Despite the lack of biochemical information, all available in silico metabolic models of Pseudomonas putida KT2440 consider NADP as the only cofactor accepted by the glucose-6-phosphate dehydrogenases. Because the Entner-Doudoroff pathway is the main glycolytic route in this bacterium, determining how much NADH and NADPH are produced in the reaction catalyzed by these enzymes is very important for the correct interpretation of metabolic flux distributions. To determine the actual cofactor preference of the glucose-6-phosphate dehydrogenase encoded by the zwf-1 gene (PputG6PDH-1), the major isoform during growth on glucose, we purified this protein and studied its kinetic properties. Based on simple kinetic principles, we estimated the in vivo relative production of NADH and NADPH during the oxidation of glucose-6-phosphate (G6P). Contrary to the general assumption, our calculations showed that the reaction catalyzed by PputG6PDH-1 yields around 1/3 mol of NADPH and 2/3 mol of NADH per mol of oxidized G6P. Additionally, we obtained data suggesting that the reaction catalyzed by the 6-phosphogluconate dehydrogenase is active during growth on glucose, and it also produces NADH. These results indicate that the stoichiometric matrix of in silico models of P. putida KT2440 must be corrected and highlight the importance of considering the physiological concentrations of the involved metabolites to estimate the actual proportion of NADH and NADPH produced by a dehydrogenase.
RESUMO
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 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 Cra(PP) 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 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.