Arnt: Structure And Mechanism Of The Aminoarabinose Transferase Responsible For Resistance To Polymyxin-Class Antibiotics

Polymyxins are cationic peptide antibiotics, once discarded due to toxicity concerns, that have been revived in the clinic and are widely used to treat multi-drug resistant (MDR) infections. Resistance to polymyxin-class antibiotics is a growing concern, as a result, one that can critically impair our ability to combat MDR infections.ArnT (4-amino-4-deoxy-L-arabinose transferase) is an integral lipid-to-lipid glycosyltransferase that modifies lipid A, the lipid component of bacterial lipopolysaccharide (LPS). ArnT is located in the inner membrane of Gram-negative bacteria and catalyzes the transfer of a modified arabinose moiety from undecaprenyl phosphate to lipid A. The addition of the arabinose moiety causes a charge modification of the bacterial outer membrane, limiting its interactions with cationic peptides, and enabling bacteria to develop resistance to polymyxin-class antibiotics and natural antimicrobial peptides.Here we report crystal structures of ArnT from a gram-negative bacterium, alone and in complex with the lipid carrier undecaprenyl phosphate, at 2.8 and 3.2A resolution, respectively. ArnT consists of a transmembrane domain with thirteen transmembrane helices and a periplasmic soluble domain. The overall fold is reminiscent of protein glycosyltransferases from bacteria and archaea, but ArnT possesses unique features that are related to its function as a lipid glycosyltransferase.Notably, we identify cavities for both lipidic substrates, accessible to the membrane environment, that converge at the active site. We observe a significant coil-to-helix structural transition upon binding of undecaprenyl phosphate that stabilizes the carrier lipid near the active site and expands the lipid A cavity, likely enabling subsequent binding of lipid A. Finally, we utilize a polymyxin resistance assay to investigate the role of particular residues in light of the structures, and propose a model for catalysis by ArnT family enzymes.