Compartmentalized Glucose Metabolism in Pseudomonas putida Is Controlled by the PtxS Repressor

Abstract

Metabolic flux analysis revealed that in Pseudomonas putida KT2440 about 50% of glucose taken up by the cells is channeled through the 2-ketogluconate peripheral pathway. This pathway is characterized by being compartmentalized in the cells. In fact, initial metabolism of glucose to 2-ketogluconate takes place in the periplasm through a set of reactions catalyzed by glucose dehydrogenase and gluconate dehydrogenase to yield 2-ketogluconate. This metabolite is subsequently transported to the cytoplasm, where two reactions are carried out, giving rise to 6-phosphogluconate, which enters the Entner-Doudoroff pathway. The genes for the periplasmic and cytoplasmic set of reactions are clustered in the host chromosome and grouped within two independent operons that are under the control of the PtxS regulator, which also modulates its own synthesis. Here, we show that although the two catabolic operons are induced in vivo by glucose, ketogluconate, and 2-ketogluconate, in vitro we found that only 2-ketogluconate binds to the regulator with an apparent K D (equilibrium dissociation constant) of 15 M, as determined using isothermal titration calorimetry assays. PtxS is made of two domains, a helix-turn-helix DNA-binding domain located at the N terminus and a C-terminal domain that binds the effector. Differential scanning calorimetry assays revealed that PtxS unfolds via two events characterized by melting points of 48.1°C and 57.6°C and that, in the presence of 2-ketogluconate, the unfolding of the effector binding domain occurs at a higher temperature, providing further evidence for 2-ketogluconate-PtxS interactions. Purified PtxS is a dimer that binds to the target promoters with affinities in the range of 1 to 3 M. Footprint analysis revealed that PtxS binds to an almost perfect palindrome that is present within the three promoters and whose consensus sequence is 5-TGAAACCGGTTTCA-3. This palindrome overlaps with the RNA polymerase binding site.

Studies of metabolite–protein interactions: A review

“…To examine metabolite-protein interactions, in vitro methods may use a binding assay (e.g., one based on ultrafiltration or equilibrium dialysis) to examine an interaction or to identify the chemicals that are involved in this process [9]. This approach can provide information such as the strength of the interaction, as well as the thermodynamics and kinetics of binding and possible conformational changes that occur as a result of the interaction [13–15]. Alternatively, an i n vitro study may make use of a method that directly examines the structure of a protein and a bound metabolite, such as occurs in X-ray crystallography or NMR spectroscopy [1,16–20].…”

“…Surface plasmon resonance (SPR) and calorimetry are two other methods that can provide information on the strength of protein-metabolite binding and the thermodynamics or kinetics of this interaction [13–15]. Studies based on SPR utilize an immobilized protein on a sensor chip, in which changes in the resonance energy (e.g., from binding of the protein with a target) are detected [9].…”

“…The change in this signal is related to the mass of the bound metabolites and can be used to determine the equilibrium constants for this process or, if examined over time, the association and dissociation kinetics that occur between the metabolite and protein during binding [9]. The reaction between a metabolite and protein can result in heat being absorbed or given off [9,13]. Calorimetry can be used to measure the overall enthalpy of the binding reaction between a metabolite and a protein [13].…”

“…The reaction between a metabolite and protein can result in heat being absorbed or given off [9,13]. Calorimetry can be used to measure the overall enthalpy of the binding reaction between a metabolite and a protein [13]. …”

Metabolic engineering and bio-electrochemical synthesis in Pseudomonas putida KT2440 for the production of p-hydroxybenzoate and 2-ketogluconate

“…To use these feedstocks, it is important to know the catabolism, regulations and metabolic responses. Glucose is common carbon source favored by many bacteria, and the use in fermentation and its catabolism and regulations have been extensively studied in many bacteria (Doelle et al, 1982;Shimizu, 2013) including P. putida (Daddaoua et al, 2010;Nikel et al, 2014a;Nikel et al, 2015b). In recent years, large-scale bio-production of biodiesel generates large amount of glycerol as by-product, which can be a promising and abundant carbon sources for industrial microbes (da Silva et al, 2009).…”