Mutagenesis studies of substrate recognition and catalysis in the sortase A transpeptidase from Staphylococcus aureus.
Journal Article (Journal Article)
The Staphylococcus aureus transpeptidase sortase A (SrtA) is responsible for anchoring a range of virulence- and colonization-associated proteins to the cell wall. SrtA recognizes substrates that contain a C-terminal LPXTG motif. This sequence is cleaved following the threonine, and an amide bond is formed between the threonine and the pentaglycine cross-bridge of branched lipid II. Previous studies have implicated the beta6/beta7 loop region of SrtA in LPXTG recognition but have not systematically characterized this domain. To better understand the individual roles of the residues within this loop, we performed alanine-scanning mutagenesis. Val-168 and Leu-169 were found to be important for substrate recognition, and Glu-171 was also found to be important, consistent with its hypothesized role as a Ca(2+)-binding residue. Gly-167 and Asp-170 were dispensable for catalysis, as was Gln-172. The role of Arg-197 in SrtA has been the subject of much debate. To explore its role in catalysis, we used native chemical ligation to generate semi-synthetic SrtA in which we replaced Arg-197 with citrulline, a non-ionizable analog. This change resulted in a decrease of <3-fold in k(cat)/K(m), indicating that Arg-197 utilizes a hydrogen bond, rather than an electrostatic interaction. Our results are consistent with a model for LPXTG recognition wherein the Leu-Pro sequence is recognized primarily by hydrophobic contacts with SrtA Val-168 and Leu-169, as well as a hydrogen bond from Arg-197. This model contradicts the previously proposed mechanism of binding predicted by the x-ray crystal structure of SrtA.
Full Text
Duke Authors
Cited Authors
- Bentley, ML; Lamb, EC; McCafferty, DG
Published Date
- May 2008
Published In
Volume / Issue
- 283 / 21
Start / End Page
- 14762 - 14771
PubMed ID
- 18375951
Pubmed Central ID
- PMC2386945
Electronic International Standard Serial Number (EISSN)
- 1083-351X
International Standard Serial Number (ISSN)
- 0021-9258
Digital Object Identifier (DOI)
- 10.1074/jbc.m800974200
Language
- eng