Structural technique applied to elucidate catalytic behavior of malic enzyme

FERNANDO ZAMARREÑO; FLORIAN HABERKORN; ROSARIO LUNARI; MARCELO D. COSTABEL; MEIKE HÜDIG; GARCÍA M.; DRINCOVICH M.F.; VERÓNICA G. MAURINO; CLARISA E. ÁLVAREZ

Rosario

Congreso; V Encuentro & II Workshop de la Red Argentina de Tecnología Enzimática; 2023

Red TEz

Deciphering how small modifications could introduce desirable characteristics into enzymes without affecting its overall function is an a highly sought-after technique for modifyingproteins in several processes. Malic enzyme (ME) is an enzyme that catalyzes the oxidative decarboxylation of malate (MLT) to pyruvate, NAD(P)H, and CO2 in the presence of a divalentmetal ion. The ME family is made up of evolutionarily related enzymes, whose biochemical and structural versatility allows them to participate in a variety of metabolic processes. Inthis context, we are carrying out structural kinetic characterization of MEs with the potential to be modified for the benefit of generating a higher yield in species with agronomicimportance. In relation to this, we recently gained novel information provided by the crystal structural analysis of the photosynthetic (C4) NADP-ME of maize and sorghum, which havespecific adapted characteristics in comparison to their no-photosynthetic (non-C4) counterpart.It is well known that C4 malic enzyme have improved catalytic efficiency compared to its non-C4 counterparts. In order to understand this different kinetic behavior, MolecularDynamics simulations (MD) of C4 and non-C4 MEs complexed with NADP, malate and Mg2+ were conducted using GROMACS 2021 [1] with AMBER03 [2] force field as well as synergiccomplementary studies of fluorescence and biochemical catalytic characterization. The integrative analysis of our results show that a different relative positioning of malate and NADPin the catalytic site of C4 and noC4 MEs was found. This analysis could provide information on how to improve these non-specialized isoforms, which are also found in C3 species such asrice, wheat, and other important agronomical crops. In the near future, direct rational design mutagenesis applied to these isoforms could be performed in order to optimize speciesthat have not yet reached their maximum potential