Estudo teórico de mecanismos para a etapa de eliminação da síntese do 5-enolpiruvilchiquimato-3-fosfato (EPSP) catalisada pela EPSP sintase de Oryza sativa

Abstract

In the sixth step of the shikimate pathway, catalyzed by 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), phosphoenolpyruvyl (PEP) reacts with shikimate-3-phosphate (S3P) to produce 5-enolpyruvylshikimate-3-phosphate (EPSP) and inorganic phosphate. Because the shikimate pathway is absent in animals, but is essential for plants and microrganisms, EPSPS is an interesting target for the development of efficient and safer herbicides, fungicides and bactericides. The most used herbicide in the world, glyphosate, is an EPSPS inhibitor. Literature results indicate that the EPSPS-catalyzed mechanism is composed by an addition step, which leads to a tetrahedral intermediate (TI), followed by an elimination step. However, the enzymatic mechanism is not completely understood, because experimental data obtained by different groups lead to conflicting results. In the present work, the EPSPScatalyzed reaction was studied by theoretical methods. First, an evaluation of the semiempirical method PM3 as a tool for the assignment of protonation states of histidine and lysine residues in enzymes was implemented by comparison with neutron diffraction data. The method was then applied for the assignment of lysine and histidine protonation states in EPSPS. Next, a model of the EPSPS of a plant, Oryza sativa, was constructed by means of homology modeling. The model was constructed based on the primary sequence of O. sativa EPSPS with two Escherichia coli EPSPS crystallographic structures as templates. After model construction, computational simulations of the elimination step were implemented with all active site aminoacid residues and water molecules, including the solvent effect. The results are indicative that the most probable mechanism for this reaction step involves the participation of a lysine residue (Lys432) as an acid catalyst and also of an aspartate residue (Asp334), as a basic catalyst. Additional studies with a phosphorylhydrazone synthesized by our group, which presented inhibitory activity for seed development, are indicative that the compound could interact very favorably with the O. sativa EPSPS active site.