Enzymatic Synthesis of Lipid II and Analogues

Huang, Li-Hsin; Huang, Shih-Hsien; Chang, Ya‐Chih; Cheng, Wei‐Chieh; Cheng, Ting‐Jen R.; Wong, Chi‐Huey

doi:10.1002/ange.201402313

Cited by 12 publications

(18 citation statements)

References 37 publications

(11 reference statements)

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“…We could further extend previous studies with the in vitro expressed Mur enzymes that were only able to reconstitute the reaction catalyzing the formation of the UDP-MurNAc-pentapeptide precursor but lacking the synthesis of the final lipid I and lipid II products by the integral membrane protein MraY and the membrane-associated protein MurG (45). Moreover in comparison to previous approaches (42) we present a fast twostep method for lipid II synthesis, which in addition does not need extensive enzyme preparations and facilitates the engineering of the biosynthetic proteins. A complete cell wall precursor biosynthesis platform could help to develop throughput assays for drug identification and the analysis of individual MraY homologues from pathogenic organisms could detect species-specific characteristics.…”

Section: Discussionmentioning

confidence: 66%

“…We achieved the reconstitution of the basic lipid II biosynthesis pathway after production of all essential proteins by cell-free expression. The modulation and detailed analysis of this pathway with enzymes originating from different pathogenic bacteria appears to become feasible (42). We could further extend previous studies with the in vitro expressed Mur enzymes that were only able to reconstitute the reaction catalyzing the formation of the UDP-MurNAc-pentapeptide precursor but lacking the synthesis of the final lipid I and lipid II products by the integral membrane protein MraY and the membrane-associated protein MurG (45).…”

Section: Discussionmentioning

confidence: 87%

“…There are further variations in the content of D-amino acids. However, it is not clear which determinants of the precursor structure are mandatory for MraY recognition, as at least some enzymes appear to be rather nonselective in terms of the sugar-modified pentapeptide (16) as well as the lipid carrier molecule (42).…”

Section: Discussionmentioning

confidence: 99%

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Lipid Requirements for the Enzymatic Activity of MraY Translocases and in Vitro Reconstitution of the Lipid II Synthesis Pathway

Henrich

Engels

et al. 2016

Journal of Biological Chemistry

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Screening of new compounds directed against key protein targets must continually keep pace with emerging antibiotic resistances. Although periplasmic enzymes of bacterial cell wall biosynthesis have been among the first drug targets, compounds directed against the membrane-integrated catalysts are hardly available. A promising future target is the integral membrane protein MraY catalyzing the first membrane associated step within the cytoplasmic pathway of bacterial peptidoglycan biosynthesis. However, the expression of most MraY homologues in cellular expression systems is challenging and limits biochemical analysis. We report the efficient production of MraY homologues from various human pathogens by synthetic cellfree expression approaches and their subsequent characterization. MraY homologues originating from Bordetella pertussis, Helicobacter pylori, Chlamydia pneumoniae, Borrelia burgdorferi, and Escherichia coli as well as Bacillus subtilis were cotranslationally solubilized using either detergent micelles or preformed nanodiscs assembled with defined membranes. All MraY enzymes originating from Gram-negative bacteria were sensitive to detergents and required nanodiscs containing negatively charged lipids for obtaining a stable and functionally folded conformation. In contrast, the Gram-positive B. subtilis MraY not only tolerates detergent but is also less specific for its lipid environment. The MraY⅐nanodisc complexes were able to reconstitute a complete in vitro lipid I and lipid II forming pipeline in combination with the cell-free expressed soluble enzymes MurA-F and with the membrane-associated protein MurG. As a proof of principle for future screening platforms, we demonstrate the inhibition of the in vitro lipid II biosynthesis with the specific inhibitors fosfomycin, feglymycin, and tunicamycin.Proteins involved in bacterial cell wall biosynthesis are highly conserved and often essential. In stage I, the cytoplasmic proteins MurA-F are responsible for the formation of the soluble peptidoglycan precursor (Park's nucleotide). In stage II, the integral membrane protein MraY catalyzes the ligation of Park's nucleotide with the membrane-bound undecaprenylphosphate (C 55 -P) 4 resulting into lipid I. The membrane-associated globular protein MurG catalyzes subsequent glycosyl transfer forming lipid II (Fig. 1). As the final monomeric building block in bacterial cell wall biosynthesis, lipid II will be transported through the membrane into the periplasmic space for further polymerization in stage III of cell wall biosynthesis (1).The bacterial cell wall biosynthetic pathway is an important and traditional target for antibiotics and numerous drugs directed against key catalysts for clinical applications have been developed. However, most known drugs such as the penicillin derivatives affect the periplasmic or extracellular steps of peptidoglycan synthesis catalyzed by soluble enzymes. In contrast, almost no clinical drugs have been developed that are targeted against the integral membrane proteins involve...

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Section: Discussionmentioning

confidence: 66%

Section: Discussionmentioning

confidence: 87%

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Lipid Requirements for the Enzymatic Activity of MraY Translocases and in Vitro Reconstitution of the Lipid II Synthesis Pathway

Henrich

Engels

et al. 2016

Journal of Biological Chemistry

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“…Chemical, chemoenzymatic, and biosynthetic routes to Lipid II variants have been developed, 13 , 14 , 18 – 27 but all are laborious. Moreover, each approach was developed for a specific Lipid II variant and considerable reengineering of the routes is required to obtain other Lipid II variants.…”

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confidence: 99%

Lipid II overproduction allows direct assay of transpeptidase inhibition by β-lactams

et al. 2017

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Peptidoglycan is an essential crosslinked polymer that surrounds bacteria and protects them from osmotic lysis. Beta-lactam antibiotics target the final stages of peptidoglycan biosynthesis by inhibiting the transpeptidases that crosslink glycan strands to complete cell wall assembly. Characterization of transpeptidases and their inhibition by beta-lactams has been hampered by lack of access to substrate. We describe a general approach to accumulate Lipid II in bacteria and to obtain large quantities of this cell wall precursor. We demonstrate utility by isolating Staphylococcus aureus Lipid II and reconstituting the synthesis of crosslinked peptidoglycan by the essential penicillin-binding protein 2, PBP2, which catalyzes both glycan polymerization and transpeptidation. We also show that we can compare the potencies of different beta-lactams by directly monitoring transpeptidase inhibition. The methods reported here will enable a better understanding of cell wall biosynthesis and facilitate studies of next-generation transpeptidase inhibitors.

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“…Other MraY studies (10,11) have also reported the use of C35-P instead of the natural lipid substrate C55-P. Polyprenyl phosphates with shorter chain lengths are also accepted as substrates by MraY (12), but to the best of our knowledge, the exact effect of the prenyl chain length on the activity of MraY has not yet been studied in detail. Together with kinetic studies, we built a model for MraY from Bacillus subtilis, carried out docking experiments with C35-P, and analyzed the conserved residues in both Gram-positive and Gram-negative MraY species.…”

mentioning

confidence: 99%

New Insight into the Catalytic Mechanism of Bacterial MraY from Enzyme Kinetics and Docking Studies

Liu

Rodrigues²,

Bonvin³

et al. 2016

Journal of Biological Chemistry

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Phospho-MurNAc-pentapeptide translocase (MraY) catalyzes the synthesis of Lipid I, a bacterial peptidoglycan precursor. As such, MraY is essential for bacterial survival and therefore is an ideal target for developing novel antibiotics. However, the understanding of its catalytic mechanism, despite the recently determined crystal structure, remains limited. In the present study, the kinetic properties of Bacillus subtilis MraY (BsMraY) were investigated by fluorescence enhancement using dansylated UDP-MurNAc-pentapeptide and heptaprenyl phosphate (C35-P, shortchain homolog of undecaprenyl phosphate, the endogenous substrate of MraY) as second substrate. Varying the concentrations of both of these substrates and fitting the kinetics data to two-substrate models showed that the concomitant binding of both UDPMurNAc-pentapeptide-DNS and C35-P to the enzyme is required before the release of the two products, Lipid I and UMP. We built a model of BsMraY and performed docking studies with the substrate C35-P to further deepen our understanding of how MraY accommodates this lipid substrate. Based on these modeling studies, a novel catalytic role was put forward for a fully conserved histidine residue in MraY (His-289 in BsMraY), which has been experimentally confirmed to be essential for MraY activity. Using the current model of BsMraY, we propose that a small conformational change is necessary to relocate the His-289 residue, such that the translocase reaction can proceed via a nucleophilic attack of the phosphate moiety of C35-P on bound UDP-MurNAc-pentapeptide.Among the enzymes involved in bacterial peptidoglycan synthesis, phospho-N-acetylmuramyl-pentapeptide translocase (MraY 4 ; EC 2.7.8.13) has been studied extensively (1, 2). This enzyme performs the initial membrane step in this process, forming undecaprenyl-N-acetylmuramyl-pentapeptide (Lipid I) from UDP-N-acetylmuramyl-pentapeptide (UDP-MurNAcpentapeptide) and undecaprenyl phosphate, in both Gram-positive and Gram-negative bacteria. Given the role of MraY in bacterial cell wall synthesis (3, 4) and cell growth (5), this enzyme is an interesting target for antibacterial drugs. Recently, the crystal structure of MraY from the Gram-negative species Aquifex aeolicus (Protein Data Bank entry 4J72) was determined (6). The enzyme was extracted from its membrane environment with detergent and crystallized as a symmetrical homodimer. Each protomer consists of 10 transmembrane helices, with both the N and C termini locating on the periplasmic side (outside) of the cytoplasmic membrane (1). Before the publication of this high resolution (3.3 Å) structure, other studies attempted to unravel the catalytic mechanism of action of MraY by site-directed mutagenesis and kinetics studies, using either membrane-embedded MraY or detergent-extracted and purified preparations (1,7,8). These studies proposed that catalysis proceeds most likely via a one-step process, although a two-step process has also been suggested (1). In the single-step process, a ternary complex of MraY, UDP-M...

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Enzymatic Synthesis of Lipid II and Analogues

Cited by 12 publications

References 37 publications

Lipid Requirements for the Enzymatic Activity of MraY Translocases and in Vitro Reconstitution of the Lipid II Synthesis Pathway

Lipid Requirements for the Enzymatic Activity of MraY Translocases and in Vitro Reconstitution of the Lipid II Synthesis Pathway

Lipid II overproduction allows direct assay of transpeptidase inhibition by β-lactams

New Insight into the Catalytic Mechanism of Bacterial MraY from Enzyme Kinetics and Docking Studies

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