Mutagenesis studies of substrate recognition and catalysis in the sortase A transpeptidase from Staphylococcus aureus

被引:63
作者
Bentley, Matthew L. [2 ,3 ]
Lamb, Erin C. [1 ]
McCafferty, Dewey G. [1 ,2 ,3 ]
机构
[1] Duke Univ, Dept Chem, Durham, NC 27708 USA
[2] Univ Penn, Sch Med, Dept Biochem & Biophys, Philadelphia, PA 19104 USA
[3] Univ Penn, Sch Med, Johnson Res Fdn, Philadelphia, PA 19104 USA
关键词
D O I
10.1074/jbc.M800974200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 LPXTGmotif. 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 beta 6/beta 7 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 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.
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收藏
页码:14762 / 14771
页数:10
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