, 2011) are critical for each of their corresponding sortase activities. When two other residues (Leu263 and Thr265) in this motif were changed to Ala, the effect on the enzyme activity was minimal (Fig. 3). Regarding whether these two residues are important for sortase activity, there are no mutagenesis data available for comparison in other pilus-related sortases. However, in the nonpilus-related SrtA, the corresponding L181A mutation has modest effect on catalytic efficiency, while T183A mutation resulted in a

1200-fold decrease in kcat relative to wild-type SrtA (Frankel et al., 2007). Because our polymerization assay only indicates the presence or the absence of activity, not the rate of the enzymatic activity, quantitative methods

need to be developed to address the effect of these mutations more precisely. To this end, our dot-blot E7080 results did show that there are less FimP Selleck ERK inhibitor components on the surface of T265A mutant than on the surface of the wild-type strain (Fig. S1). It has been proposed that sortases use a catalytic triad composed of His-Cys-Arg during the catalytic process (Table 3). The His204 residue is most likely the His residue in the catalytic triad. The His 204 residue is located 6 Å from the Cys 266 SG atom (Persson, 2011). The H204A mutation effect is consistent with what has been reported about other histidine residues located in the catalytic triads. For instance, in pilus-related sortases, its counterparts His 131in SrtC-1 of S. pneumoniae and His157 in SrtC1 of Group

B Streptococcus were essential for pilus fiber formation in both organisms (Manzano et al., 2009; Cozzi et al., 2011). In the nonpilus-related SrtA from S. aureus, His120 residue at a similar location in relation to the essential Cys184 residue is also critical for the catalytic process (Ilangovan et al., 2001; Ton-That et al., 2002). However, the catalytic function of this critical histidine residue is still a subject of debate. It is speculated that the His residue, because it is being positively charged, might contribute to the electrostatic environment essential for the catalytic activity (Zong et al., 2004). Although the newly published crystal structure (Persson, 2011) showed that Arg275 is part of the His-Cys-Arg catalytic triad, our results for indicate that this Arginine residue is not important for the SrtC1 activity in A. oris T14V. In contrast, its counterparts Arg202 in SrtC-1 of S. pneumoniae and Arg228 in SrtC1 of Group B Streptococcus are essential for the activity of the corresponding sortases (Manzano et al., 2009; Cozzi et al., 2011). Even in the nonpilus-related sortase SrtA, the Arginine 197 residue was identified to be important for the enzyme’s activity(Frankel et al., 2007). However, in SrtA, the Arg197 residue is 13 amino acids away from the essential Cys194 residue instead of the nine-amino acid distance between the arginine and cysteine residues in the SrtC catalytic triads.