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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.

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.