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 ␤6/␤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 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.The increasing prevalence of multidrug-resistant Gram-positive bacterial infections has been a cause for great concern (1, 2). Most alarming has been the recent characterization of strains of Staphylococcus aureus which exhibit either intermediate or full resistance to vancomycin, currently the antibiotic of last resort for methicillin-resistant S. aureus infections (3-5). During pathogenesis, Gram-positive bacteria use virulence factors to aid in adherence to host endothelial tissues, evasion of the immune system, and the acquisition of iron from the local environment (6, 7). There has been a great deal of recent interest in the idea of targeting these virulence factors as a possible strategy for the development of new therapeutics for the treatment of 9).Sortases are cysteine transpeptidase enzymes found in a wide-range of Gram-positive pathogens. They catalyze the formation of a covalent linkage between surface proteins containing an LPXTG motif and the peptidoglycan layer of the cell wall (6). Many of these cell wall-anchored proteins function as virulence factors (6, 10). As such, several previous studies have implicated a role for sortases in promoting virulence in several strains of Gram-positive bacteria. Sortase knock-out strains have been shown to have reduced pathogenicity in S. aureus (11), Listeria monocytogenes (12, 13), and in several members of the Streptococcus lineage: S. gordonii (14), S. pneumonia (15), S. pyoge...

Three naturally occurring pyrrole acids were found in Sepia, human black hair, and bovine choroid and iris melanosomes using high-performance liquid chromatography and mass spectrometry--pyrrole-2,3-dicarboxylic acid (PDCA), pyrrole-2,3,5-tricarboxylic acid (PTCA) and pyrrole-2,3,4,5-tetracarboxylic acid (PTeCA). PDCA and PTCA are common markers quantified from oxidative degradation of eumelanins. Using standards, the amounts of naturally occurring PDCA and PTCA were determined and compared to those obtained following peroxide oxidation of the same samples. Because the naturally occurring acids are water soluble, these results indicate that care must be exercised when comparing PDCA and PTCA yields from the degradation analyses of melanins isolated and prepared by different methods. This work also establishes that PTeCA is a naturally occurring pyrrole acid in melanosomes.