Resistance to the “last resort” antibiotic colistin: a single-zinc mechanism for phosphointermediate formation in MCR enzymes

Lythell, Emily; Suardíaz, Reynier; Hinchliffe, P.; Hanpaibool, Chonnikan; Visitsatthawong, Surawit; Oliveira, A. Sofia F.; Lang, Eric J. M.; Surawatanawong, Panida; Lee, Vannajan Sanghiran; Rungrotmongkol, Thanyada; Fey, Natalie; Spencer, James; Mulholland, Adrian J.

doi:10.1039/d0cc02520h

Cited by 14 publications

(37 citation statements)

References 25 publications

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“…7,8 It was previously demonstrated that the first step of the reaction was not sensitive to the exact ε value used. 6 Here, we show that the same applies for the second step of the reaction (Table S4 †). Since typically the effect of ε on DFT cluster model calculations saturates with increasing system size, 29 we consider the relatively small cluster used here to be appropriate for this system.…”

Section: Communicationsupporting

confidence: 64%

Catalytic mechanism of the colistin resistance protein MCR-1

et al. 2021

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Section: Communicationsupporting

confidence: 64%

Catalytic mechanism of the colistin resistance protein MCR-1

et al. 2021

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“…7,8 It was previously demonstrated that the first step of the reaction was not sensitive to the exact ε value used. 6 Here, we show that the same applies for the second step of the reaction (Table S4). Since typically the effect of ε on DFT cluster model calculations saturates with increasing system size, 28 we consider the relatively small cluster used here to be appropriate in size.…”

Section: Cluster Model and Calculationssupporting

confidence: 62%

“…Possible mechanisms for PEA transfer to the Thr285 acceptor, the first of the two-step reaction mechanism, have previously been studied using DFT cluster model calculations. 5 , 6 Here we explored an expanded range of alternative mechanisms, assessed these using higher levels of theory, and extended the study to the whole reaction. Exploratory studies of the different potential reaction pathways and different choices of protonation states for the histidine residues were carried out using semiempirical PM6 Hamiltonian and B3LYP/6-31G(d) levels of theory before being submitted to B3LYP-GD3BJ calculations and the previously described combination of basis sets (see SI for a detailed description and Tables S1 for results).…”

Section: Phosphoethanolamine Transfer To the Proteinmentioning

confidence: 99%

“…5 Those calculations suggested that MCR-1 can support PEA transfer to Thr285 with only one Zn 2+ ion bound. 6 Here, we have used a cluster model of the MCR-1 active site to model, for the first time, both steps of catalytic mechanism using DFT and ab initio calculations. Cluster models have been used successfully to study enzyme mechanisms for many years, especially for metallo-proteins.…”

Section: Introductionmentioning

confidence: 99%

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Catalytic Mechanism of the Colistin Resistance Protein MCR-1

Suardíaz

Lythell²,

Hinchliffe³

et al. 2020

Preprint

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<div> <div> <div> <p>The mcr-1 gene encodes a membrane-bound Zn2+-metalloenzyme, MCR-1, which catalyzes 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 trans-fer 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 involment 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. </p> </div> </div> </div>

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“…2 The problem of AMR is particularly urgent given the alarming proliferation of antibiotic resistance in bacteria; pathogens associated with both community-acquired and healthcare-associated infections are increasingly resistant to first-line and even reserve agents. 3 This not only poses a serious challenge obstacle in fighting common and severe bacterial infections, but also reduces the viability and increases the risks of interventions such as orthopedic surgery and also threatens new antibiotics coming to the market. 4 AMR risks negating a century of progress in medicine made possible by the ability to effectively treat bacterial infections.…”

Section: Introductionmentioning

confidence: 99%

Allosteric communication in Class A β-lactamases occurs via Cooperative Coupling of Loop Dynamics

Galdadas

Oliveira

et al. 2021

Preprint

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Allosteric effects control protein (e.g. enzyme) activity in ways that are not fully understood. Better understanding of allosteric effects, and tools to identify them, would offer promising alternative strategies to inhibitor development. Through a combination of equilibrium and nonequilibrium molecular dynamics simulations, we identify allosteric effects and communication pathways from two distant ligand binding sites to important active site structural elements that control enzymatic activity in two prototypical class A β-lactamases, TEM-1 and KPC-2. Both of these enzymes are important determinants of antibiotic resistance in widespread bacterial pathogens. The simulations show that the allosteric sites are connected to the active site in both enzymes, (e.g. affecting the conformation of the Ω-loop) highlighting how allosteric inhibitors may exert their effects. Nonequilibrium simulations reveal pathways of communication operating over distances of 30 Å or more. In these identified signaling pathways, the propagation of the signal occurs through cooperative coupling of loop dynamics. Notably, 50% or more clinically relevant amino acid substitutions in each enzyme map onto the identified signal transduction pathways. This suggests that clinically important variation may affect, or be driven by, differences in allosteric behavior, providing a mechanism by which amino acid substitutions may affect the relationship between spectrum of activity, catalytic turnover and potential allosteric behavior in this clinically important enzyme family. Simulations of the type presented here will help in identifying and analyzing such differences.

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Resistance to the “last resort” antibiotic colistin: a single-zinc mechanism for phosphointermediate formation in MCR enzymes

Abstract: Simulations show the mono-zinc form of MCR to be stable and competent for covalent phospho(ethanolamine) intermediate formation.

Cited by 14 publications

References 25 publications

Catalytic mechanism of the colistin resistance protein MCR-1

Catalytic mechanism of the colistin resistance protein MCR-1

Catalytic Mechanism of the Colistin Resistance Protein MCR-1

Allosteric communication in Class A β-lactamases occurs via Cooperative Coupling of Loop Dynamics

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