Cloning, purification, and characterization of the 6-phosphogluconate dehydrogenase (6 PGDH) from Giardia lamblia

• Published in: Molecular and biochemical parasitology 2021-07, Vol.244, p.111383-111383, Article 111383
• Main Authors: Morales-Luna, Laura, Hernández-Ochoa, Beatriz, Martínez-Rosas, Víctor, González-Valdez, Abigail, Cárdenas-Rodríguez, Noemi, Enríquez-Flores, Sergio, Marcial-Quino, Jaime, Gómez-Manzo, Saúl
• Format: Article
• Language: English
• Subjects: 6PGDH protein; Cloning; Homology modeling; Kinetic parameters; Purification
• ISSN: 0166-6851; 1872-9428
• DOI: 10.1016/j.molbiopara.2021.111383

•Functional cloning of Gl-6 PGDH was performed.•The Gl-6 PGDH enzyme was purified by affinity column to determine the biochemical and physicochemical properties.•Enzymatic characterization of recombinant 6 PGDH exhibiting similar properties with other 6 PGDHs parasites.•Homology modeling of Gl-6 PGDH has shown low similarity with respect to the structure of the human 6 PGDH protein. Giardia lamblia, due to the habitat in which it develops, requires a continuous supply of intermediate compounds that allow it to survive in the host. The pentose phosphate pathway (PPP) provides essential molecules such as NADPH and ribulose-5-phosphate during the oxidative phase of the pathway. One of the key enzymes during this stage is 6-phosphogluconate dehydrogenase (6 PGDH) for generating NADPH. Given the relevance of the enzyme, in the present work, the 6pgdh gene from G. lamblia was amplified and cloned to produce the recombinant protein (Gl-6 PGDH) and characterize it functionally and structurally after the purification of Gl-6 PGDH by affinity chromatography. The results of the characterization showed that the protein has a molecular mass of 54 kDa, with an optimal pH of 7.0 and a temperature of 36–42 °C. The kinetic parameters of Gl-6 PGDH were Km = 49.2 and 139.9 μM (for NADP+ and 6-PG, respectively), Vmax =26.27 μmol*min−1*mg−1, and Kcat = 24.0 s−1. Finally, computational modeling studies were performed to obtain a structural visualization of the Gl-6 PGDH protein. The generation of the model and the characterization assays will allow us to expand our knowledge for future studies of the function of the protein in the metabolism of the parasite.