Antibiotic resistance is an increasing global concern threatening our ability to fight bacterial infections. A key part of the innate immune response is Cationic Antimicrobial Peptides (CAMPs) that constitute an effective defense barrier against bacterial colonization. Colistin is a cyclic CAMP antibiotic from the polymyxin family used as a last-resort antibiotic against Multi Drug Resistant Gram-negative bacteria. Alarmingly, reports of resistance to colistin are increasingly common. Covalent modification of lipid A with 4-deoxy-4-amino-L-arabinose (Ara4N) mediates resistance to CAMPs and colistin in Gram-negative bacteria. The proteins required for Ara4N biosynthesis are encoded in the pmrE and arnBCADTEF loci, with ArnT ultimately transferring the amino sugar from undecaprenyl-phospho-4-deoxy-4- amino-L-arabinose (C55P-Ara4N) to lipid A. However, Ara4N is N-formylated prior to its transfer to undecaprenyl-phosphate by ArnC while ArnD is thought to mediate the deformylation. This thesis provides insights into the structural and biochemical properties of both ArnC and ArnD. A low-resolution map of ArnC solubilized in detergent shows a tetrameric architecture consistent with members of the glycosyltransferase family. ArnC reconstituted into nanodiscs and imaged by cryoEM displays diverse 2D-classes providing a path towards a high resolution Cryo-EM structure. The crystal structure of ArnD is presented, from which five specific motifs are characterized suggesting that ArnD represents a new family of carbohydrate esterases. The active site presents a metal coordination H-H-D triad and, in the presence of Co2+ or Mn2+, ArnD efficiently deformylates C55P-Ara4FN confirming its role in Ara4N biosynthesis. The reconstitution of both ArnC and ArnD activities in vitro is explored for assay development and diprenyl derivatives (neryl-phosphate) of the undecaprenyl substrates, were confirmed to work as a substrate for both enzymes. Having appropriate assays to test ArnC and ArnD enzyme activity is essential for the evaluation of potential inhibitors of the Arn4N modification of lipid A unlocking new avenues for testing lead compounds for clinical development in the fight against polymyxin resistance.</p
