Design and synthesis of FMN derivatives for covalent binding to Anabaena apoflavodoxin

Abstract

Resumen del trabajo presentado a la XI National Conference BIFI, celebrada del 25 al 27 de enero de 2023 en el Campus Río Ebro, Zaragoza (España).Many flavoproteins are involved in vital metabolic transformations, which are of high biotechnological relevance and play essential biological roles in small organisms and humans. Flavodoxins are electron- transfer flavoproteins that contain one molecule of non-covalently bound flavin mononucleotide as the redox component. This flavoprotein is subject to dissociation equilibrium and influenced by solution conditions, which may result in cofactor dissociation, leading to destabilization of the protein moiety and to irreversible loss of its catalytic activity. In order to develop a new strategy for the rational increase of flavoproteins conformational stability, we have been focused on getting variants of the Anabaena flavodoxin bearing covalently bound catalytically active FMN; for which we have synthesized two FMN derivatives which contain electrophilic and reactive groups. Suitable flavodoxin variants have also been designed, expressed and purified. We have performed a brief computational study to confirm that these compounds are suitable to establish covalent links with new flavodoxin mutants and that the links are compatible with maintaining the native orientation of the isoalloxacin moiety relative to the apoprotein. Currently, we are carrying out experiments to find the best conditions to achieve such covalent binding of FMN derivatives through nucleophilic residues strategically located near the active center.Peer reviewe