Our simulations also allowed for investigation of interactions of MCR-1, in both mono-and di-zinc forms and in the presence and absence of PEtN-modified Thr285, with the POPE donor substrate. Notably, in all cases POPE remained associated with MCR-1, due in large part to interactions of the lipid tail with the transmembrane domain. However, close approach of the POPE head group to the active center was possible only in simulations involving the "resting" form of the enzyme, i.e. the mono-zinc form with unmodified Thr285. Under these conditions, POPE, which initially sampled orientations in which the PEtN primary amine contacted the conserved "gating" histidine residues His395 and His478, was (in one of three replicate simulations) able to adopt a stable conformation compatible with subsequent reaction with Thr285, i.e. with the PEtN phosphorous atom approaching O of the Thr285 acceptor. Importantly, this position was not reached in simulations of di-zinc MCR-1, in which the POPE phosphoryl oxygen groups preferentially interacted with the Zn2 ion, or in simulations of the mono-zinc enzyme with Thr285 modified by covalent addition of PEtN, where interaction of the PEtN amine with His395 and/or His478 prevented further entry of POPE into the active site. Taken together with others' experimental observations that the "resting" as-isolated state of the purified recombinant enzyme contains a single zinc equivalent , these data then support mechanistic proposals wherein the first stage of the PEtN transfer reaction, i.e. addition of PEtN to Thr285, involves reaction of the donor substrate (e.g. POPE) with the mono-, rather than di-zinc, form of the enzyme.