Structures of aminoarabinose transferase ArnT suggest a molecular basis for lipid A glycosylation

Petrou, Vasileios I.; Herrera, Carmen M.; Schultz, Kathryn M.; Clarke, Oliver; Vendôme, Jérémie; Tomasek, David; Banerjee, Surajit; Rajashankar, Kanagalaghatta R.; Dufrisne, Meagan Belcher; Kloss, Brian; Kloppmann, Edda; Rost, Burkhard; Klug, Candice S.; Trent, M. Stephen; Shapiro, Lawrence; Mancia, Filippo

doi:10.1126/science.aad1172

Cited by 94 publications

(113 citation statements)

References 46 publications

(43 reference statements)

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“…Unlike the chromosome-encoded enzyme ArnT which features 11 N-terminal transmembrane regions (38), the MCR-1 protein is an integral membrane protein with five periplasmic transmembrane helices (12, 13). In light of the lack of significant homology (27.1% similarity and 12.7% identity) between the two enzymes (ArnT and MCR-1), we are very interested in examining in the near future whether or not they are functionally replaced in E. coli .…”

Section: Discussionmentioning

confidence: 99%

Deciphering MCR-2 Colistin Resistance

Sun

Gao

et al. 2017

mBio

124

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Antibiotic resistance is a prevalent problem in public health worldwide. In general, the carbapenem β-lactam antibiotics are considered a final resort against lethal infections by multidrug-resistant bacteria. Colistin is a cationic polypeptide antibiotic and acts as the last line of defense for treatment of carbapenem-resistant bacteria. Very recently, a new plasmid-borne colistin resistance gene, mcr-2, was revealed soon after the discovery of the paradigm gene mcr-1, which has disseminated globally. However, the molecular mechanisms for MCR-2 colistin resistance are poorly understood. Here we show a unique transposon unit that facilitates the acquisition and transfer of mcr-2. Evolutionary analyses suggested that both MCR-2 and MCR-1 might be traced to their cousin phosphoethanolamine (PEA) lipid A transferase from a known polymyxin producer, Paenibacillus. Transcriptional analyses showed that the level of mcr-2 transcripts is relatively higher than that of mcr-1. Genetic deletions revealed that the transmembrane regions (TM1 and TM2) of both MCR-1 and MCR-2 are critical for their location and function in bacterial periplasm, and domain swapping indicated that the TM2 is more efficient than TM1. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) confirmed that all four MCR proteins (MCR-1, MCR-2, and two chimeric versions [TM1-MCR-2 and TM2-MCR-1]) can catalyze chemical modification of lipid A moiety anchored on lipopolysaccharide (LPS) with the addition of phosphoethanolamine to the phosphate group at the 4′ position of the sugar. Structure-guided site-directed mutagenesis defined an essential 6-residue-requiring zinc-binding/catalytic motif for MCR-2 colistin resistance. The results further our mechanistic understanding of transferable colistin resistance, providing clues to improve clinical therapeutics targeting severe infections by MCR-2-containing pathogens.

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Section: Discussionmentioning

confidence: 99%

Deciphering MCR-2 Colistin Resistance

Sun

Gao

et al. 2017

mBio

124

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“…Recently, the structure of the lipid-to-lipid glycosyltransferase 4-amino-4-deoxy-L-arabinose transferase (ArnT) has been determined (35). ArnT is an integral membrane bound glycosyltransferase that attaches the carbohydrate, 4-amino-4-deoxy-L-arabinose (L-Ara4N) to the 1 and 4′ phosphate groups of lipid A, the same site of modification for EptA.…”

Section: Resultsmentioning

confidence: 99%

Structure of a lipid A phosphoethanolamine transferase suggests how conformational changes govern substrate binding

Anandan

Evans

Čondić‐Jurkić

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

115

192

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Multidrug-resistant (MDR) gram-negative bacteria have increased the prevalence of fatal sepsis in modern times. Colistin is a cationic antimicrobial peptide (CAMP) antibiotic that permeabilizes the bacterial outer membrane (OM) and has been used to treat these infections. The OM outer leaflet is comprised of endotoxin containing lipid A, which can be modified to increase resistance to CAMPs and prevent clearance by the innate immune response. One type of lipid A modification involves the addition of phosphoethanolamine to the 1 and 4′ headgroup positions by phosphoethanolamine transferases. Previous structural work on a truncated form of this enzyme suggested that the full-length protein was required for correct lipid substrate binding and catalysis. We now report the crystal structure of a full-length lipid A phosphoethanolamine transferase from Neisseria meningitidis, determined to 2.75-Å resolution. The structure reveals a previously uncharacterized helical membrane domain and a periplasmic facing soluble domain. The domains are linked by a helix that runs along the membrane surface interacting with the phospholipid head groups. Two helices located in a periplasmic loop between two transmembrane helices contain conserved charged residues and are implicated in substrate binding. Intrinsic fluorescence, limited proteolysis, and molecular dynamics studies suggest the protein may sample different conformational states to enable the binding of two very different-sized lipid substrates. These results provide insights into the mechanism of endotoxin modification and will aid a structure-guided rational drug design approach to treating multidrug-resistant bacterial infections.lipid modification | multidrug resistance | molecular dynamics | Neisseria | membrane protein structure

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“…In bacteria, GT activity is required for virulence and viability, and individual GTs are emerging as novel targets for antimicrobial and anti-virulence drug discovery [2,3]. Small molecular inhibitors of GTs are therefore sought after as chemical tools for the interrogation, manipulation and disruption of cellular glycosylation pathways [4], and as potential lead compounds for drug discovery.…”

Section: Introductionmentioning

confidence: 99%

Covalent inhibitors of LgtC: A blueprint for the discovery of non-substrate-like inhibitors for bacterial glycosyltransferases

Smith

Vivoli

et al. 2017

Bioorganic & Medicinal Chemistry

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. Covalent inhibitors of LgtC: a blueprint for the discovery of non-substrate-like inhibitors for bacterial glycosyltransferases. Bioorganic and Medicinal Chemistry, 25(12), 3182-3194. https://doi.org/10.1016/j.bmc.2017.04.006Citing this paper Please note that where the full-text provided on King's Research Portal is the Author Accepted Manuscript or Post-Print version this may differ from the final Published version. If citing, it is advised that you check and use the publisher's definitive version for pagination, volume/issue, and date of publication details. And where the final published version is provided on the Research Portal, if citing you are again advised to check the publisher's website for any subsequent corrections. General rightsCopyright and moral rights for the publications made accessible in the Research Portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognize and abide by the legal requirements associated with these rights.•Users may download and print one copy of any publication from the Research Portal for the purpose of private study or research.•You may not further distribute the material or use it for any profit-making activity or commercial gain •You may freely distribute the URL identifying the publication in the Research Portal Take down policyIf you believe that this document breaches copyright please contact librarypure@kcl.ac.uk providing details, and we will remove access to the work immediately and investigate your claim. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.-1 - data reveals that non-catalytic cysteines are a common motif in the active site of many bacterial glycosyltransferases. Our results can therefore serve as a blueprint for the rational design of non-substrate-like, covalent inhibitors against a broad range of other bacterial glycosyltransferases.-3 -

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Structures of aminoarabinose transferase ArnT suggest a molecular basis for lipid A glycosylation

Cited by 94 publications

References 46 publications

Deciphering MCR-2 Colistin Resistance

Deciphering MCR-2 Colistin Resistance

Structure of a lipid A phosphoethanolamine transferase suggests how conformational changes govern substrate binding

Covalent inhibitors of LgtC: A blueprint for the discovery of non-substrate-like inhibitors for bacterial glycosyltransferases

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