Deciphering allosteric properties of a malate metabolizing enzyme through in silico analysis and site-directed mutagenesis

GERRARD WHEELER, M. C.; ARIAS, C. L.; DA FONSECA REZENDE E MELLO, J.; CIRAUQI DIAZ, N.; RANGEL RODRIGUES, C.; DRINCOVICH, M. F.; MENDONÇA TELES DE SOUZA, A.; ALVAREZ, C. E.

Evento virtual

Workshop; Primer Workshop de la Red Argentina de Tecnología Enzimática; 2021

Red Argentina de Tecnología Enzimática

Enzyme function could be integratively analyzed by structural and biochemical studies in order to design and introduce desired characteristics. Malic enzyme (ME) is a ubiquitous enzyme that catalyzes the oxidative decarboxylation of malate. The high number of ME crystal structures available from different species facilitates comparisons between sequence, structure, and function. Here we focus in the study of structural determinants necessary for fumarate allosteric activation of the NADP-ME2 isoform from Arabidopsis thaliana. As the 3D structure for this enzyme is not yet reported, we characterized the NADP-ME2 allosteric site by structural modeling, molecular docking, normal mode analysis, and mutagenesis. Our results suggest that other C4 acids including malate, NADP-ME2 substrate, could also fit into fumarate?s pocket, extending the modulation sensing capacity. A new group of non-conserved hydrophobic residues was identified in the second sphere adjacent to the allosteric site. The substitution of one of those residues, L62, by a less flexible residue as tryptophan, resulted in a complete loss of fumarate activation and a reduction of substrate affinities for the active site. In addition, conformational changes leading to the regulation could originate in the region surrounding L62, extending through the allosteric site till the active site. Altogether these results contribute to the understanding about how minimal differences in the amino acid sequence impact on the regulatory signal transmission into the enzyme without affecting the global protein structure.