ABSTRACT
The major autolysins (Atl) of Staphylococcus epidermidis and S. aureus play an important role in cell separation, and their mutants are also attenuated in virulence. Therefore, autolysins represent a promising target for the development of new types of antibiotics. Here, we report the high-resolution structure of the catalytically active amidase domain AmiE (amidase S. epidermidis) from the major autolysin of S. epidermidis. This is the first protein structure with an amidase-like fold from a bacterium with a gram-positive cell wall architecture. AmiE adopts a globular fold, with several alpha-helices surrounding a central beta-sheet. Sequence comparison reveals a cluster of conserved amino acids that define a putative binding site with a buried zinc ion. Mutations of key residues in the putative active site result in loss of activity, enabling us to propose a catalytic mechanism. We also identified and synthesized muramyltripeptide, the minimal peptidoglycan fragment that can be used as a substrate by the enzyme. Molecular docking and digestion assays with muramyltripeptide derivatives allow us to identify key determinants of ligand binding. This results in a plausible model of interaction of this ligand not only for AmiE, but also for other PGN-hydrolases that share the same fold. As AmiE active-site mutations also show a severe growth defect, our findings provide an excellent platform for the design of specific inhibitors that target staphylococcal cell separation and can thereby prevent growth of this pathogen.
Subject(s)
Amidohydrolases , N-Acetylmuramoyl-L-alanine Amidase , Staphylococcus epidermidis/growth & development , Staphylococcus epidermidis/genetics , Acetylmuramyl-Alanyl-Isoglutamine/metabolism , Amidohydrolases/chemistry , Amidohydrolases/genetics , Amidohydrolases/metabolism , Amino Acid Sequence , Binding Sites/physiology , Catalytic Domain , Cell Wall/physiology , Crystallography , Enzyme Activation/physiology , Molecular Sequence Data , Mutagenesis , N-Acetylmuramoyl-L-alanine Amidase/chemistry , N-Acetylmuramoyl-L-alanine Amidase/genetics , N-Acetylmuramoyl-L-alanine Amidase/metabolism , Protein Folding , Protein Structure, Tertiary , Staphylococcus epidermidis/pathogenicity , Structure-Activity Relationship , Substrate Specificity , VirulenceABSTRACT
The bifunctional Autolysin E from Staphylococcus epidermidis, contains a Zn(2+)-dependent N-acetylmuramoyl-L-alanine amidase AmiE (EC 3.5.1.28). This enzyme hydrolyzes the amide bond between the carbohydrate chain and the peptide stem of bacterial peptidoglycan. Since peptidoglycan is the mayor component of bacterial cell walls, type II amidases like Autolysin E play an essential role in the bacterial life cycle. Therefore bacterial amidases are appropriate drug targets in the development of antibiotics. The drug discovery process relies on sensitive enzyme assay systems to test possible lead candidates for enzyme inhibition. However, specific determination of bacterial amidase activity is complicated because a simple and accurate enzyme assay is currently unavailable. In this study we developed a sensitive fluorescent substrate for the type II amidase Autolysin E from S. epidermidis, which is suitable for quantifying amidase activity in a high throughput compatible fashion. Using derivatives of the substrate Mca-Ala-D-isoGln-Lys(Dnp)-D-Ala-Arg-OH, we were further able to characterize the amidase substrate specificity of Autolysin E.
