Mutagenesis Studies of Substrate Recognition and Catalysis in the Sortase A Transpeptidase from Staphylococcus aureus

• Published in: The Journal of biological chemistry Vol. 283; no. 21; pp. 14762 - 14771
• Main Authors: Bentley, Matthew L., Lamb, Erin C., McCafferty, Dewey G.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 23.05.2008; American Society for Biochemistry and Molecular Biology
• Subjects: Aminoacyltransferases - chemistry; Aminoacyltransferases - genetics; Aminoacyltransferases - metabolism; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Binding Sites; Catalysis; Crystallography, X-Ray; Cysteine Endopeptidases - chemistry; Cysteine Endopeptidases - genetics; Cysteine Endopeptidases - metabolism; Enzyme Catalysis and Regulation; Kinetics; Models, Molecular; Mutagenesis - genetics; Mutation - genetics; Peptidyl Transferases - chemistry; Peptidyl Transferases - genetics; Peptidyl Transferases - metabolism; Protein Denaturation; Protein Structure, Tertiary; Staphylococcus aureus; Staphylococcus aureus - enzymology; Staphylococcus aureus - genetics; Substrate Specificity; Temperature
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M800974200

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 β6/β7 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 Ca2+-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 kcat/Km, 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.