Catalytic mechanism of MraY and WecA, two paralogues of the polyprenyl-phosphate N-acetylhexosamine 1-phosphate transferase superfamily

Al-Dabbagh, Bayan; Olatunji, Samir; Crouvoisier, Muriel; Ghachi, Meriem El; Blanot, Didier; Mengin‐Lecreulx, Dominique; Bouhss, Ahmed

doi:10.1016/j.biochi.2016.06.005

Cited by 41 publications

(56 citation statements)

References 37 publications

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“…This bacterial polyisoprenoid anchors the building blocks for peptidoglycan and LPS synthesis to the inner leaflet of the membrane and enables flippases to shuttle them across the membrane. The MraY transferase synthesizing lipid I (Al-Dabbagh et al, 2016) is present in all species that have complete or intermediate peptidoglycan, and absent in all those lacking peptidoglycan (Fig. 4D).…”

Section: Biosynthesis Of Peptidoglycan Precursorsmentioning

confidence: 99%

Peptidoglycan in obligate intracellular bacteria

Otten

Brilli

Vollmer

et al. 2017

Molecular Microbiology

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SummaryPeptidoglycan is the predominant stress‐bearing structure in the cell envelope of most bacteria, and also a potent stimulator of the eukaryotic immune system. Obligate intracellular bacteria replicate exclusively within the interior of living cells, an osmotically protected niche. Under these conditions peptidoglycan is not necessarily needed to maintain the integrity of the bacterial cell. Moreover, the presence of peptidoglycan puts bacteria at risk of detection and destruction by host peptidoglycan recognition factors and downstream effectors. This has resulted in a selective pressure and opportunity to reduce the levels of peptidoglycan. In this review we have analysed the occurrence of genes involved in peptidoglycan metabolism across the major obligate intracellular bacterial species. From this comparative analysis, we have identified a group of predicted ‘peptidoglycan‐intermediate’ organisms that includes the Chlamydiae, Orientia tsutsugamushi, Wolbachia and Anaplasma marginale. This grouping is likely to reflect biological differences in their infection cycle compared with peptidoglycan‐negative obligate intracellular bacteria such as Ehrlichia and Anaplasma phagocytophilum, as well as obligate intracellular bacteria with classical peptidoglycan such as Coxiella, Buchnera and members of the Rickettsia genus. The signature gene set of the peptidoglycan‐intermediate group reveals insights into minimal enzymatic requirements for building a peptidoglycan‐like sacculus and/or division septum.

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Section: Biosynthesis Of Peptidoglycan Precursorsmentioning

confidence: 99%

Peptidoglycan in obligate intracellular bacteria

Otten

Brilli

Vollmer

et al. 2017

Molecular Microbiology

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“…This locates the diphosphate group close to the catalytic residues D93, D94 and K111 (ref. 18) and D231 involved in Mg 2+ ion binding 15 . The N-GlcNAc ring partially overlaps with the tunicamycin counterpart, while the position of the L-Ala indicates the direction of the substrate pentapeptide, interacting with helix 9c and the neighboring loops, similarly to the MD2 peptide ( Supplementary Fig.…”

mentioning

confidence: 99%

“…L112 is conserved as a hydrophobic and/or large residue in MraYs, while G166 is conserved as a small residue, suggesting that the shape and properties of this region are important for activity. An entry path of the C 55 P between helices 4, 5 and 9 is complicated by the reported lack of lipid competitivity of tunicamycin 12 , but would be in line with the one-step MraY mechanism in which D93 deprotonates one of the C 55 P hydroxyls that makes a nucleophilic attack on the β-phosphate of UDP-MurNAc to form the lipid I product 17,18 .…”

mentioning

confidence: 99%

MraY–antibiotic complex reveals details of tunicamycin mode of action

Hakulinen¹,

Hering²,

Brändén³

et al. 2017

Nat Chem Biol

107

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The rapid increase of antibiotic resistance has created an urgent need to develop novel antimicrobial agents. Here we describe the crystal structure of the promising bacterial target phospho-N-acetylmuramoyl-pentapeptide translocase (MraY) in complex with the nucleoside antibiotic tunicamycin. The structure not only reveals the mode of action of several related natural-product antibiotics but also gives an indication on the binding mode of the MraY UDP-MurNAc-pentapeptide and undecaprenyl-phosphate substrates.

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“…In contrast, WecA-catalyzed transformation of 1 with cis -C 10 -P or cis -C 15 -P or α- cis -geranylgeranyl phosphate ( cis -C 20 -P) did not provide the corresponding prenyl-P-P-Glucosamine-C 6 -FITC derivatives, but generated C 50 -P-P-Glucosamine-C 6 -FITC in 5–10% yield, whose reactions were achieved by using endogenous decaprenyl phosphate (C 50 -P) (Fig 5). Thus, it was concluded that the WecA enzymes are specific to the prenyl substrate; in our experiments, all truncated prenyl phosphates including α- trans prenyl phosphates failed to react with 1 (43,51). …”

Section: Discussionmentioning

confidence: 52%

“…through the Michaelis-Menten plot. Significant difference in WecA-catalyzed phosphoryltransfer reactions is that the transformation between UDP-Glucosamine-C 6 -FITC ( 1 ) and C 50 -P-P-Glucosamine-C 6 -FITC ( 3 ) is not a reverse process, whereas the recent studies have concluded that Thermotoga maritima WecA-catalyzed reaction shows equilibrium between UDP-GlcNAc and C 55 -P-P-GlcNAc (51). Similarly, MraY/MurX-catalyzed phosphoryltransfer of polyprenyl phosphate to Park’s nucleotide is known to be an equilibrium reaction (31).…”

Section: Resultsmentioning

confidence: 99%

Fluorescence-based assay for polyprenyl phosphate-GlcNAc-1-phosphate transferase (WecA) and identification of novel antimycobacterial WecA inhibitors

Mitachi

Siricilla

Yang

et al. 2016

Analytical Biochemistry

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Polyprenyl phosphate-GlcNAc-1-phosphate transferase (WecA) is an essential enzyme for the growth of Mycobacterium tuberculosis (Mtb) and some other bacteria. Mtb WecA catalyzes the transformation from UDP-GlcNAc to decaprenyl-P-P-GlcNAc, the first membrane-anchored glycophospholipid that is responsible for the biosynthesis of mycolylarabinogalactan in Mtb. Inhibition of WecA will block the entire biosynthesis of essential cell wall components of Mtb in both replicating and non-replicating states, making this enzyme a target for development of novel drugs. Here, we report a fluorescence-based method for the assay of WecA using a modified UDP-GlcNAc, UDP-Glucosamine-C6-FITC (1), a membrane fraction prepared from an M. smegmatis strain, and the E. coli B21WecA. Under the optimized conditions, UDP-Glucosamine-C6-FITC (1) can be converted to the corresponding decaprenyl-P-P-Glucosamine-C6-FITC (3) in 61.5% yield. Decaprenyl-P-P-Glucosamine-C6-FITC is readily extracted with n-butanol and can be quantified by ultraviolet-visible (UV-Vis) spectrometry. Screening of the compound libraries designed for bacterial phosphotransferases resulted in the discovery of a selective WecA inhibitor, UT-01320 (12) that kills both replicating and non-replicating Mtb at low concentration. UT-01320 (12) also kills the intracellular Mtb in macrophages. We conclude that the WecA assay reported here is amenable to medium- and high-throughput screening, thus facilitating the discovery of novel WecA inhibitors.

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Catalytic mechanism of MraY and WecA, two paralogues of the polyprenyl-phosphate N-acetylhexosamine 1-phosphate transferase superfamily

Cited by 41 publications

References 37 publications

Peptidoglycan in obligate intracellular bacteria

Peptidoglycan in obligate intracellular bacteria

MraY–antibiotic complex reveals details of tunicamycin mode of action

Fluorescence-based assay for polyprenyl phosphate-GlcNAc-1-phosphate transferase (WecA) and identification of novel antimycobacterial WecA inhibitors

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