Abstract
We report the first direct observation of the self-association behavior of the Staphylococcus aureus sortase A (SrtA) transpeptidase. Formation of a SrtA dimer was observed under native conditions by polyacrylamide gel electrophoresis and fast protein liquid chromatography (FPLC). Subsequent peptide mass fingerprinting and protein sequencing experiments confirmed the dimeric form of the SrtA protein. Furthermore, SrtA can be selectively cross-linked both in vitro and in Escherichia coli. Multiple samples of enzyme were subjected to analytical sedimentation equilibrium ultracentrifugation to obtain an apparent Kd for dimer formation of about 55 microM. Finally, enzyme kinetic studies suggested that the dimeric form of SrtA is more active than the monomeric enzyme. Discovery of SrtA dimerization may have significant implications for understanding microbial physiology and developing new antibiotics.
Publication types
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Comparative Study
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Research Support, Non-U.S. Gov't
MeSH terms
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Aminoacyltransferases / biosynthesis
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Aminoacyltransferases / chemistry*
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Aminoacyltransferases / genetics
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Aminoacyltransferases / isolation & purification
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Bacterial Proteins / biosynthesis
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Bacterial Proteins / chemistry*
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Bacterial Proteins / genetics
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Bacterial Proteins / isolation & purification
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Cloning, Molecular
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Cross-Linking Reagents / chemistry
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Cysteine Endopeptidases / biosynthesis
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Cysteine Endopeptidases / chemistry*
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Cysteine Endopeptidases / genetics
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Cysteine Endopeptidases / isolation & purification
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Dimerization
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Escherichia coli / enzymology
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Escherichia coli / genetics
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Gene Expression Regulation, Bacterial
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Kinetics
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Peptide Mapping
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Peptidoglycan Glycosyltransferase / chemistry
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Peptidoglycan Glycosyltransferase / genetics
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Peptidoglycan Glycosyltransferase / isolation & purification
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Sequence Analysis, Protein
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Staphylococcus aureus / enzymology*
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Staphylococcus aureus / genetics
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Ultracentrifugation
Substances
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Bacterial Proteins
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Cross-Linking Reagents
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Aminoacyltransferases
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sortase A
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Peptidoglycan Glycosyltransferase
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Cysteine Endopeptidases