Overall mutational scanning unveils the essential active residues for the mechanistic action of MCR-1.

Polymyxins, including colistin and polymyxin B, serve as crucial last-resort antibiotics for managing infections caused by carbapenem-resistant Enterobacterales (CRE). However, the rapid spread of the mobilized colistin resistance gene (mcr-1) challenged the efficacy of treatment by polymyxins. The mcr-1 gene encoded a transmembrane phosphoethanolamine (PEA) transferase enzyme, MCR-1. MCR-1 could catalyze the transfer of PEA moiety of phosphatidylethanolamine (PE) to the 1' (or 4')-phosphate group of the lipid A. Despite the determination of several structures of the soluble domain of MCR-1, the structural and biochemical mechanisms of integral MCR-1 remain less understood. In this study, we utilized an alanine scanning mutagenesis approach to systematically investigate the functional attributes of distinct regions within MCR-1. We identified fifteen critical residues that are indispensable for the enzymatic activity of MCR-1 and are pivotal for its ability to confer resistance to colistin. Furthermore, molecular docking of MCR-1 complexed with the phosphoethanolamine (PE) substrate revealed the presence of a channel-shaped cavity, a characteristic feature shared with other phosphoethanolamine transferases. Despite MCR-1 exhibiting a low sequence identity with both MCR homologues and other phosphoethanolamine (PEA) transferases, several conserved sites were identified, including Y, M, K, H, L, and H, suggesting a potentially shared catalytic mechanism among them for modifying LPS-lipid A. Overall, these findings provide a deep understanding of the catalytic mechanism of MCR-1 for colistin resistance. Moreover, these findings provide a robust structural and functional foundation, enabling the rational design of targeted inhibitors and restoring colistin activity against serious infections with carbapenem-resistant Enterobacterales (CRE).