Development of screening assays and discovery of initial inhibitors of pneumococcal peptidoglycan deacetylase PgdA

Bui, Nhat Khai; Turk, Samo; Buckenmaier, Stephan; Stevenson-Jones, Flint; Zeuch, Benjamin; Gobec, Stanislav; Vollmer, Waldemar

doi:10.1016/j.bcp.2011.03.028

Cited by 32 publications

(17 citation statements)

References 27 publications

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“…The S. pneumoniae PgdA protein ( sp PdgA), L. monocytogenes PgdA (lmo0415) and L. lactis PgdA (XynD) were identified as N-acetylglucosamine deacetylase proteins containing CE-4 NodB domains [10,25,26]. Rv1096 also contained a CE-4 NodB domain.…”

Section: Resultsmentioning

confidence: 99%

Mycobacterium tuberculosis Rv1096 protein: gene cloning, protein expression, and peptidoglycan deacetylase activity

et al. 2014

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BackgroundMany bacteria modulate and evade the immune defenses of their hosts through peptidoglycan (PG) deacetylation. The PG deacetylases from Streptococcus pneumonia, Listeria monocytogenes and Lactococcus lactis have been characterized. However, thus far, the PG deacetylase of Mycobacterium tuberculosis has not been identified.ResultsIn this study, we cloned the Rv1096 gene from the M. tuberculosis H37Rv strain and expressed Rv1096 protein in both Escherichia coli and M. smegmatis. The results showed that the purified Rv1096 protein possessed metallo-dependent PG deacetylase activity, which increased in the presence of Co2+. The kinetic parameters of the PG deacetylase towards M. smegmatis PG as a substrate were as follows: Km, 0.910 ± 0.007 mM; Vmax, 0.514 ± 0.038 μMmin-1; and Kcat = 0.099 ± 0.007 (S-1). Additionally, the viability of M. smegmatis in the presence of over-expressed Rv1096 protein was 109-fold higher than that of wild-type M. smegmatis after lysozyme treatment. Additionally, light microscopy and scanning electron microscopy showed that in the presence of over-expressed Rv1096 protein, M. smegmatis kept its regular shape, with an undamaged cell wall and smooth surface. These results indicate that Rv1096 caused deacetylation of cell wall PG, leading to lysozyme resistance in M. smegmatis.ConclusionWe have determined that M. tuberculosis Rv1096 is a PG deacetylase. The PG deacetylase activity of Rv1096 contributed to lysozyme resistance in M. smegmatis. Our findings suggest that deacetylation of cell wall PG may be involved in evasion of host immune defenses by M. tuberculosis.

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Section: Resultsmentioning

confidence: 99%

Mycobacterium tuberculosis Rv1096 protein: gene cloning, protein expression, and peptidoglycan deacetylase activity

et al. 2014

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“…The fact that the enzyme is localized to and functions outside of the cell makes it all the more attractive as an antibacterial target. Interestingly, another peptidoglycan deacetylase, the peptidoglycan N ‐acetylglucosamine deacetylase PgdA that is confined to Gram‐positive species, has also been targeted for antibiotic development and the screening for novel inhibitors against this activity was recently reported 25…”

Section: Resultsmentioning

confidence: 99%

Identification of the First Known Inhibitors of O‐Acetylpeptidoglycan Esterase: A Potential New Antibacterial Target

Pfeffer

Clarke

2012

ChemBioChem

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The O-acetylation of peptidoglycan (PG) is now known to occur in 53 species, including numerous human pathogens such as, Staphylococcus aureus, Bacillus anthracis, species of Enterococcus, Campylobacter jejuni, Helicobacter pylori, Neisseria gonorrhoeae and N. meningitidis. This modification, which occurs at the C-6 hydroxyl of N-acetylmuramoyl residues within PG, serves to regulate autolytic activity during PG metabolism and contributes to pathogenesis and persistence within a host. O-Acetylpeptidoglycan esterase (Ape) was recently discovered as an enzyme responsible for the removal of O-acetyl groups from PG, thus permitting the continued maintenance and metabolism of the sacculus. Recombinant Ape1 from N. gonorrhoeae was purified to apparent homogeneity and optimal storage conditions for the enzyme were determined. Using 4-methylumbelliferyl acetate as substrate, a fluorogenic assay amenable for the high-throughput screening for potential inhibitors was developed and Ape1 was screened against a subset of compounds of the Canadian Compound Collection. The overall Z' score for the screen was 0.62, indicative of a well-suited assay with a sufficient signal window, and the threshold was set at 65 %. After eliminating a number of false-positives, seven compounds were identified as true inhibitors of Ape1, the first to be identified for this class of enzyme. Dose-response curves were generated leading to the identification of five of these compounds with IC(50) values ranging between 0.3 and 23 μM. Of these, purpurin was selected for further analysis and it was found to inhibit the growth of both Gram-positive and Gram-negative bacteria that produce both O-acetylated PG and Ape.

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“…CE4 enzymes are capable of removing acetyl groups in chitin, chitosan, and CHOS, thus converting GlcNAc (or A) units to GlcN (or D) units. Enzymes in the CE4 family may also act on peptidoglycan [ 7 , 8 ] and acetyl xylan [ 9 ]. The use of CDAs could in principle allow tailoring of both the fraction and pattern of acetylation in chitosan and CHOS [ 10 – 12 ].…”

Section: Introductionmentioning

confidence: 99%

Structure and function of a CE4 deacetylase isolated from a marine environment

et al. 2017

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Chitin, a polymer of β(1–4)-linked N-acetylglucosamine found in e.g. arthropods, is a valuable resource that may be used to produce chitosan and chitooligosaccharides, two compounds with considerable industrial and biomedical potential. Deacetylating enzymes may be used to tailor the properties of chitin and its derived products. Here, we describe a novel CE4 enzyme originating from a marine Arthrobacter species (ArCE4A). Crystal structures of this novel deacetylase were determined, with and without bound chitobiose [(GlcNAc)2], and refined to 2.1 Å and 1.6 Å, respectively. In-depth biochemical characterization showed that ArCE4A has broad substrate specificity, with higher activity against longer oligosaccharides. Mass spectrometry-based sequencing of reaction products generated from a fully acetylated pentamer showed that internal sugars are more prone to deacetylation than the ends. These enzyme properties are discussed in the light of the structure of the enzyme-ligand complex, which adds valuable information to our still rather limited knowledge on enzyme-substrate interactions in the CE4 family.

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Development of screening assays and discovery of initial inhibitors of pneumococcal peptidoglycan deacetylase PgdA

Cited by 32 publications

References 27 publications

Mycobacterium tuberculosis Rv1096 protein: gene cloning, protein expression, and peptidoglycan deacetylase activity

Mycobacterium tuberculosis Rv1096 protein: gene cloning, protein expression, and peptidoglycan deacetylase activity

Identification of the First Known Inhibitors of O‐Acetylpeptidoglycan Esterase: A Potential New Antibacterial Target

Structure and function of a CE4 deacetylase isolated from a marine environment

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