Quantifying NAD(P)H production in the upper Entner-Doudoroff pathway from Pseudomonas putida KT2440

Author(s)

Olavarria, Karel ^[1] ; Marone, Marina Pupke ^[1] ; Oliveira, Henrique da Costa ^[1] ; Roncallo, Juan Camilo ^[1] ; da Costa Vasconcelos, Fernanda Nogales ^[1] ; da Silva, Luiziana Ferreira ^[1] ; Cabrera Gomez, Jose Gregrio ^[1]

Affiliation

^[1] Univ Sao Paulo, Inst Ciencias Biomed, Dept Microbiol, BR-05508 Sao Paulo - Brazil

• Document type: Journal article
• Source: FEBS OPEN BIO; v. 5, p. 908-915, 2015.
• Web of Science Citations: 1

Abstract

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. (C) 2015 The Authors. Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. This is an open access article under the CC BY-NC-ND license. (AU)

• FAPESP's process: 13/50357-2 - Programmable balancing of growth and formation of poyhydroxyalkanoates in Escherichia coli
• Grantee: José Gregório Cabrera Gomez
• Support type: Regular Research Grants
• FAPESP's process: 13/24087-8 - Metabolic impact of transient genetic perturbations in Escherichia coli and Pseudomonas putida
• Grantee: Karel Olavarria Gamez
• Support type: Scholarships in Brazil - Post-Doctorate