Pseudomonas putida KT2440 Strain Metabolizes Glucose through a Cycle Formed by Enzymes of the Entner-Doudoroff, Embden-Meyerhof-Parnas, and Pentose Phosphate Pathways

Pablo I Nikel,Max Chavarría,Tobias Fuhrer,Uwe Sauer,Víctor De Lorenzo

doi:10.1074/jbc.m115.687749

Abstract

The soil bacterium Pseudomonas putida KT2440 lacks a functional Embden-Meyerhof-Parnas (EMP) pathway, and glycolysis is known to proceed almost exclusively through the Entner-Doudoroff (ED) route. To investigate the raison d'être of this metabolic arrangement, the distribution of periplasmic and cytoplasmic carbon fluxes was studied in glucose cultures of this bacterium by using (13)C-labeled substrates, combined with quantitative physiology experiments, metabolite quantification, and in vitro enzymatic assays under both saturating and non-saturating, quasi in vivo conditions. Metabolic flux analysis demonstrated that 90% of the consumed sugar was converted into gluconate, entering central carbon metabolism as 6-phosphogluconate and further channeled into the ED pathway. Remarkably, about 10% of the triose phosphates were found to be recycled back to form hexose phosphates. This set of reactions merges activities belonging to the ED, the EMP (operating in a gluconeogenic fashion), and the pentose phosphate pathways to form an unforeseen metabolic architecture (EDEMP cycle). Determination of the NADPH balance revealed that the default metabolic state of P. putida KT2440 is characterized by a slight catabolic overproduction of reducing power. Cells growing on glucose thus run a biochemical cycle that favors NADPH formation. Because NADPH is required not only for anabolic functions but also for counteracting different types of environmental stress, such a cyclic operation may contribute to the physiological heftiness of this bacterium in its natural habitats.

Highlights

Glucose metabolism in many bacteria is based on the standard linear Embden-Meyerhof-Parnas pathway
Growth of P. putida KT2440 Mutants Lacking Key Enzymes of the Central Carbon Metabolism—The starting point of this study was the assessment of the relevance of the three glucose catabolism routes in P. putida KT2440, composed of the ED, PP, and the partial EMP pathways (Fig. 1 and supplemental Table S3)
The EMP pathway used by many organisms is considered to be the predominant textbook route for metabolism of glucose [16, 56], in reality the ED counterpart is the most frequent biochemical device found in free-living bacteria and archaea

Summary

Background

Glucose metabolism in many bacteria is based on the standard linear Embden-Meyerhof-Parnas pathway. Our results demonstrate that the ED pathway merges its activity with a gluconeogenic operation of the upper EMP and the PP pathway for recycling triose phosphates back into hexose phosphates This situation gives rise to a metabolic itinerary of key intermediates through what we call the EDEMP cycle (i.e. recruiting activities from the ED, EMP, and PP pathways). This particular metabolic architecture could have evolved to ensure an appropriate supply of NADPH reducing power for coping with the environmental stress that prevails in the natural niches of this bacterium

Experimental Procedures

Results

Discussion