Catalytic mechanism of the colistin resistance protein MCR-1

Abstract

The mcr-1 gene encodes a membrane-bound Zn2+-metalloenzyme, MCR-1, which catalyses phosphoethanolamine transfer onto bacterial lipid A, making bacteria resistant to colistin, a last-resort antibiotic. Mechanistic understanding of this process remains incomplete. Here, we investigate possible catalytic pathways using DFT and ab initio calculations on cluster models and identify a complete two-step reaction mechanism. The first step, formation of a covalent phosphointermediate via transfer of phosphoethanolamine from a membrane phospholipid donor to the acceptor Thr285, is rate-limiting and proceeds with a single Zn2+ ion. The second step, transfer of the phosphoethanolamine group to lipid A, requires an additional Zn2+. The calculations suggest the involvement of the Zn2+ orbitals directly in the reaction is limited, with the second Zn2+ acting to bind incoming lipid A and direct phosphoethanolamine addition. The new level of mechanistic detail obtained here, which distinguishes these enzymes from other phosphotransferases, will aid in the development of inhibitors specific to MCR-1 and related bacterial phosphoethanolamine transferases.

Fig. 1. Model of the MCR-1 enzyme (cyan, cartoon) based upon MCR-1 catalytic domain crystal structure and the full-length N. meningitidis EptA structure shown embedded in bacterial inner membrane (coloured spheres). Mono-Zn active site with PEA-donor phospholipid substrate in coloured sticks. Only polar protons are shown.

Fig. 2. First step of the reaction: phosphoethanolamine transfer to the protein. Stationary points of the proposed reaction pathway are shown in 3D as sticks (top, only selected protons shown, transferring protons in white spheres) and in 2D (bottom). (A) Reactant state. (B) Transition state, concerted transfer of two protons and formation and cleavage of P–O bonds. (C) Product state before substrate departure. Zn-ligand coordination distances indicated in black.

Fig. 3. Second step of the reaction: PEA transfer to the lipid A. Stationary points of the proposed reaction pathway are shown in 3D as sticks (top, only selected protons shown, transferring proton in white sphere) and in 2D (bottom). (A) Reactant state after substrate 1 departure and lipid A binding. (B) Transition state, concerted return of proton transferred in the first step and formation/cleavage of P–O bonds. (C) Product state before substrate departure. Zn-ligand coordination distances indicated in black.