JOURNAL ARTICLE

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

Matthew L Bentley, Erin C Lamb, Dewey G McCafferty
Journal of Biological Chemistry 2008 May 23, 283 (21): 14762-71
18375951
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 Links

Find Full Text Links for this Article

Discussion

You are not logged in. Sign Up or Log In to join the discussion.

Trending Papers

Remove bar
Read by QxMD icon Read
18375951
×

Save your favorite articles in one place with a free QxMD account.

×

Search Tips

Use Boolean operators: AND/OR

diabetic AND foot
diabetes OR diabetic

Exclude a word using the 'minus' sign

Virchow -triad

Use Parentheses

water AND (cup OR glass)

Add an asterisk (*) at end of a word to include word stems

Neuro* will search for Neurology, Neuroscientist, Neurological, and so on

Use quotes to search for an exact phrase

"primary prevention of cancer"
(heart or cardiac or cardio*) AND arrest -"American Heart Association"