Tracking transpeptidase activities in vitro
Bacterial peptidoglycan (PG) synthesis requires transpeptidase activity to construct covalent cross-linkages between glycan strands through polypeptides. Transpeptidases are valuable antibacterial targets. Their inactivation results in failed cell wall synthesis and leads to cell death. In vitro assays to study transpeptidation activity have been developed previously and have greatly advanced our understanding of PG synthesis mechanisms. [1] However, due to the presence of strong background noise from the leftover probes, signal output in the existing transpeptidase assays can only be obtained after product purification processes. This complicates the experiments and increases the amount of human work required. Given that rotor-fluorogenic D-amino acids (RfDAAs) fluoresce upon PG incorporation, a continuous, spectrophotometric assay to monitor transpeptidation reactions without the need for product purification has been reported recently. [2]
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In this assay, RfDAA was employed as the fluorescence reporter and synthetic diacetyl-l-lysine-d-alanine-d-alanine was used as the substrate (acyl donor) to examine Staphylococcus aureus penicillin-binding protein 4 (PBP4) activity. Coupling to the substrates increased the steric hindrance of RfDAA. This resulted in an increased fluorescence intensity which was detected by a plate reader. S. aureus PBP4 was chosen here because it is a known D,D-transpeptidase responsible for PG cross-linking in the staphylococci, as well as a β-lactamase that degrades penicillin-like antibiotics. Knocking out PBP4 results in significantly decreased resistance to β-lactams in methicillin-resistant S. aureus (MRSA), making it a valuable target for new antibiotic development. [3]
When this assay was initiated by adding the enzyme of the probe and substrate mixture, an increase in intensity was observed. The formation of the cross-linked product was confirmed by HPLC and highresolution mass spectrometry. Control experiments using the L-enantiomer of Rf470DL showed no signal increase in the assay, which is in agreement with the stereocenter selectivity of PBPs. To confirm that the reaction was carried out by enzyme activity, a known effective inhibitor of S. aureus PBP4, cefoxitin, was added to the reaction. Total inhibition of D,D-transpeptidation activity was observed. In contrast, when chloramphenicol, a ribosome peptidyl transferase inhibitor, was added, no inhibition effect was found. These results indicate that the signal increase in the assay results from S. aureus PBP4 activity and that RfDAAs could be employed for real-time monitoring of the D,D-transpeptidation reaction in vitro.
References
[1] Qiao et al. Lipid II overproduction allows direct assay of transpeptidase inhibition by β-lactams. Nat. Chem. Biol. 2017, 13, 793–798.
[2] Hsu et al. Fluorogenic D-amino acids enable real-time monitoring of peptidoglycan biosynthesis and high-throughput transpeptidation assays. Nat Chem. 2019, 11 (4):335-341
[3] Navratna et al. Molecular basis for the role of Staphylococcus aureus penicillin-binding protein 4 in antimicrobial resistance. J. Bacteriol. 2010, 192, 134–144.