IAL   21557
INSTITUTO DE AGROBIOTECNOLOGIA DEL LITORAL
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
STUDY OF CELERY ENZYMES INVOLVED IN MANNITOL METABOLISM
Autor/es:
MINEN, ROMINA I.; BALLICORA, MIGUEL ÁNGEL; LIU, DALI; BHAYANI, JAINA; FIGUEROA, CARLOS M.; IGLESIAS, ALBERTO A.
Reunión:
Congreso; LVI SAIB Meeting ?XV SAMIGE Meeting; 2020
Resumen:
In celery (Apium graveolens), almost 80% of newly fixed CO2 is equally partitioned between sucrose and the sugar-alcohol mannitol (Mol), while only a minor part is allocated to starch. Besides their role as photosynthates, sugar-alcohols have a relevant function as compatible solutes, allowing plants to cope with different types of abiotic stress (cold, salinity, and drought). In this work, we characterized two enzymes involved in Mol metabolism in celery plants: mannose-6-phosphate reductase (Man6PRase, EC 1.1.1.224), which catalyzes the NADPH-dependent conversion of Man6P to Mol1P in photosynthetic tissues; and Mol dehydrogenase (MolDHase, EC 1.1.1.255), an NAD+-dependent enzyme that converts Mol into Man in heterotrophic tissues. Recombinant proteins were obtained with a His-tag at the N-terminus and purified by IMAC. Man6PRase and MolDHase are homodimers of 65 and 85 kDa, respectively. Man6PRase displayed a Vmax of 3.5 U mg-1 in the direction of Man6P reduction, with S0.5 values of 4.4 and 0.026 mM for Man6P and NADPH, respectively. MolDHase showed a Vmax of 1.8 U mg-1 for the oxidation of Mol, and S0.5 values of 25.5 and 0.97 mM for Mol and NAD+, respectively. We solved the crystal structure of Man6PRase in complex with mannonic acid and NADP+, which allowed us to determine the amino acids that are putatively involved in the binding of Man6P. Based on this structure, we rationalized that Lys48 could be important for binding the phosphate moiety of Man6P. TheMan6PRase K48A mutant?s catalytic efficiencies were 4-fold higher with Man and one order of magnitude lower with Man6P than those of the WT enzyme. To evaluate the specificity of MolDHase for its substrates, we first constructed a 3D model of the enzyme using the crystal structure of the sinapyl alcohol dehydrogenase from Populus tremuloides (1YQD) as a template. Then, we were able to identify three amino acids that would be critical to switch the cofactor specificity of MolDHase, from NAD+ to NADP+. Our results show that the catalytic efficiency of the MolDHase triple mutant (D214S-I215T-S343N) is 141-fold higher for NADP+ than for NAD+, while the catalytic efficiency of the WT with NAD+ is almost 80-fold higher than with NADP+. Overall, our work provides detailed kinetic and structural knowledge to elucidate the determinants of substrate binding and better understand the reaction mechanisms operating in the main enzymes involved in Mol metabolism. This work also lays the groundwork for obtaining new, promiscuous enzymes to expand the spectrum of molecules with different and unique applications, mainly in biorefineries.