The flavin transferase ApbE is a lipoprotein located in the periplasm of numerous bacterial species. ApbE binds to FAD noncovalently and facilitates the exclusive insertion of the FMN cofactor via a phosphoester bond to a threonine residue within the target protein [1]. Through this posttranslational modification (FMNylation), ApbE matures several bacterial proteins involved in energy metabolism, including subunits of unique multienzyme complexes: the Na+-translocating NADH: ubiquinone oxidoreductase (C and B) [2] and the Rhodobacter nitrogen fixation (RNF) complex (G and D) [3], along with other proteins like fumarate reductase and urocanate reductase [4]. As a critical assembly factor, ApbE emerges as an excellent target for drug discovery. Inhibiting the active site of ApbE could impede the maturation of Na+ -NQR and other enzymes vital for microbial cellular respiration. Our pursuit of this target in multidrug-resistant pathogens utilizes the structure-based drug discovery approach: Crystallography-based fragment screening. For this, the ApbE enzyme from E. coli was produced recombinantly and purified by affinity and size exclusion chromatographic steps under retention of catalytic activity. Crystals of ApbE, obtained through automated screening and optimized for X-ray diffraction up to 1.8 Å, revealed the FAD binding pocket. Identification of unique interactions and key residues within the 3D structure of ApbE sets the stage for the next step. Using ApbE crystals, we aim to implement a fast fragment and compound screening pipeline, providing a potential starting point for developing a new class of antibiotics to combat antimicrobial resistance, especially in multidrug-resistant gram-negative bacteria

[1] Bogachev, Baykov,and Bertsova(2018). Biochemical Society transactions 46, 5, 1161–1169.

[2] Méheust, Raphaël; Huang, Shuo; Rivera-Lugo, Rafael; Banfield, Jillian; Light, Samuel (2021) eLife 10. DOI: 10.7554/eLife.66878

[3] Hau, Kaltwasser, Muras, Casutt, Vohl, Claußen, Steffen, Leitner, Bill, Cutsail, DeBeer, Vonck, Steuber, Fritz(2023). Nat. Struct. Mol. Biol. 2023

[4] Vitt, Prinz, Eisinger, Ermler, and Buckel. (2022). Nature Communications 13,6315