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Unusual substrate specificity of the peptidoglycan MurE ligase from Erysipelothrix rhusiopathiae
Delphine Patin1, Samo Turk2, Hélène Barreteau1
1Institut de Biologie Intégrative de la Cellule (I2BC), UMR 9198 CEA/CNRS/Université Paris-Sud, 91405 Orsay, France.
Abstract:
Erysipelothrix rhusiopathiae is a Gram-positive bacterium pathogenic to many species of birds and mammals, including humans. The main feature of its peptidoglycan is the presence of l-alanine at position 3 of the peptide stem. In the present work, we cloned the murE gene from E. rhusiopathiae and purified the corresponding protein as His6-tagged form. Enzymatic assays showed that E. rhusiopathiae MurE was indeed an l-alanine-adding enzyme. Surprisingly, it was also able, although to a lesser extent, to add meso-diaminopimelic acid, the amino acid found at position 3 in many Gram-negative bacteria, Bacilli and Mycobacteria. Sequence alignment of MurE enzymes from E. rhusiopathiae and Escherichia coli revealed that the DNPR motif that is characteristic of meso-diaminopimelate-adding enzymes was replaced by HDNR. The role of the latter motif in the interaction with l-alanine and meso-diaminopimelic acid was demonstrated by site-directed mutagenesis experiments and the construction of a homology model. The overexpression of the E. rhusiopathiae murE gene in E. coli resulted in the incorporation of l-alanine at position 3 of the peptide part of peptidoglycan.
Insights
Erysipelothrix rhusiopathiae MurE enzyme primarily adds L-alanine to peptidoglycan, a key component in bacterial cell walls. This study reveals its unique dual-amino acid adding capability and the structural basis for its function.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Erysipelothrix rhusiopathiae is a Gram-positive bacterium causing disease in various species, including humans.
- The unique peptidoglycan structure of E. rhusiopathiae, with L-alanine at peptide stem position 3, is a potential target for antimicrobial development.
Purpose of the Study:
- To clone and characterize the murE gene and its protein product from E. rhusiopathiae.
- To investigate the enzymatic activity and substrate specificity of E. rhusiopathiae MurE.
- To elucidate the structural determinants of MurE's amino acid incorporation.
Main Methods:
- Cloning and expression of the E. rhusiopathiae murE gene.
- Purification of His6-tagged E. rhusiopathiae MurE protein.
- Enzymatic assays to determine amino acid incorporation.
- Sequence alignment and site-directed mutagenesis of MurE.
- Homology modeling of the MurE enzyme.
- Overexpression of E. rhusiopathiae murE in E. coli.
Main Results:
- E. rhusiopathiae MurE was successfully cloned, purified, and confirmed as an L-alanine-adding enzyme.
- The enzyme exhibited a surprising, albeit lesser, ability to incorporate meso-diaminopimelic acid.
- Sequence analysis revealed a unique HDNR motif in E. rhusiopathiae MurE, differing from the DNPR motif in E. coli MurE.
- Mutagenesis and modeling studies confirmed the role of the HDNR motif in substrate interaction.
- Overexpression of E. rhusiopathiae murE in E. coli led to L-alanine incorporation at peptidoglycan position 3.
Conclusions:
- E. rhusiopathiae MurE possesses a unique enzymatic activity, primarily adding L-alanine but also capable of incorporating meso-diaminopimelic acid.
- The HDNR motif is crucial for the substrate specificity of E. rhusiopathiae MurE.
- Understanding E. rhusiopathiae MurE function provides insights into bacterial cell wall biosynthesis and potential antimicrobial targets.
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