Mutagenesis studies of substrate recognition and catalysis in the sortase A transpeptidase from Staphylococcus aureus.
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.
Bentley
Matthew L
ML
Department of Biochemistry and Biophysics and the Johnson Research Foundation, The University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
Lamb
Erin C
EC
McCafferty
Dewey G
DG
D019881:Aminoacyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016296:Mutagenesis / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D010458:Peptidyl Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D019881:Aminoacyltransferases; C120542:sortase A; D010458:Peptidyl Transferases; D003546:Cysteine Endopeptidases