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. 2008 Jan 15;105(2):431-6.
doi: 10.1073/pnas.0710868105. Epub 2008 Jan 8.

Domain requirement of moenomycin binding to bifunctional transglycosylases and development of high-throughput discovery of antibiotics

Ting-Jen Rachel Cheng  1 , Ming-Ta Sung, Hsin-Yu Liao, Yi-Fan Chang, Chia-Wei Chen, Chia-Ying Huang, Lien-Yang Chou, Yen-Da Wu, Yin-Hsuan Chen, Yih-Shyun E Cheng, Chi-Huey Wong, Che Ma, Wei-Chieh Cheng
Affiliations

Domain requirement of moenomycin binding to bifunctional transglycosylases and development of high-throughput discovery of antibiotics

Ting-Jen Rachel Cheng et al. Proc Natl Acad Sci U S A. .

Abstract

Moenomycin inhibits bacterial growth by blocking the transglycosylase activity of class A penicillin-binding proteins (PBPs), which are key enzymes in bacterial cell wall synthesis. We compared the binding affinities of moenomycin A with various truncated PBPs by using surface plasmon resonance analysis and found that the transmembrane domain is important for moenomycin binding. Full-length class A PBPs from 16 bacterial species were produced, and their binding activities showed a correlation with the antimicrobial activity of moenomycin against Enterococcus faecalis and Staphylococcus aureus. On the basis of these findings, a fluorescence anisotropy-based high-throughput assay was developed and used successfully for identification of transglycosylase inhibitors.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Inhibition of TG by moenomycin and binding affinities of truncated PBP variants. (A) Moe A (1) inhibits the transglycosylation step in bacterial cell wall synthesis. (B) Schematic representation of PBP variants used for moenomycin binding studies. The full-length PBP (TM + TG + TP) contains amino acid residues 1–844 of the E. coli PBP1b P02919. TG + TP (residues 88–844), TM + TG (residues 1–409), TG (residues 195–409), and TP (residues 444–736) are truncated variants with that portion of the full-length PBP1b. The moenomycin binding constant (KD) for these variants was determined by using SPR.
Fig. 2.
Fig. 2.
Design of an FA assay for TG. (A and B) Chemical structures of modified Moe A (2) and fluorescein-labeled moenomycin, F-Moe (3). (C and D) Results of SPR analysis of the binding activity of Moe A (C) and F-Moe (D) to E. coli PBP1b. Shown are responses for moenomycin binding to immobilized E. coli PBP1b. The data were analyzed by using steady-state affinity and fitted to a 1:1 interaction model (Insets). The KD values deduced from the intercepts of the x axis and the dotted lines are 440 and 520 nM for Moe A and F-Moe, respectively.
Fig. 3.
Fig. 3.
Development of a high-throughput FA assay for TG. (A) Concentration-dependent changes in FA were observed when E. coli PBP1b bound to F-Moe. (B) Improved FA assay with H. pylori PBP1a. The concentration-dependent FA changes were performed similarly to A but using the H. pylori PBP1a. The maximum anisotropy value was 0.2. (C) Displacement of the PBP1a-bound F-Moe complex by unlabeled moenomycin at various concentrations. The changes in FA are defined as [(Aobs − Amin)/(Amax − Amin) × 100%]. The KD and IC50 values were calculated as described in SI Materials and Methods.
Fig. 4.
Fig. 4.
Chemical structures of moenomycin derivatives (4 and 5).
Fig. 5.
Fig. 5.
Screening for small molecules as TG inhibitors using the class A PBPs. (A) HTS for TG inhibitors using the FA assay. Protein structure graphics were created from Protein Data Bank (www.pdb.org) ID code 2OLV (12). (B) Chemical structures of the HTS hits (compounds 6–8).
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