Molecular Insights into Functional Differences Between mcr-3- and mcr-1-Mediated Colistin Resistance [PublishAheadOfPrint]

The global emergence of plasmid-mediated colistin resistance genes mcr-1 and mcr-3 has threatened the role of the "last resort" drug colistin in the defense against infections caused by multidrug-resistant Gram-negative bacteria. However, functional differences between these two genes in mediating colistin resistance remains poorly understood. Protein sequence alignment of MCR-3 and MCR-1 was therefore conducted in Clustal Omega to identify sequence divergence. The molecular recognition of lipid A head group phosphatidylethanolamine and MCR-3 enzyme was studied by homology modeling and molecular docking, with the catalytic mechanism of MCR-3 also being explored. Thr277 in MCR-3 was validated as the key amino acid residue responsible for the catalytic reaction using site-directed mutagenesis and was shown to act as a nucleophile. Lipid A modification induced by the MCR-3 and MCR-1 enzymes was confirmed by electrospray ionization time-of-flight mass spectrometry. Far-UV circular dichroism spectra of the MCR-3 and MCR-1 enzymes suggested that MCR-3 was more thermostable than MCR-1, with a melting temperature of 66.19°C compared with 61.14°C for MCR-1. These data provided molecular insight into the functional differences between mcr-3 and mcr-1 in conferring colistin resistance.

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