The surface of Gram-positive bacteria is a highly regulated environment with specific attachment of proteins required for viability. Sortase enzymes recognize and ligate substrates to the peptidoglycan layer in these microorganisms, which can be highly pathogenic (e.g., Staphylococcus aureus). As such, sortases represent a potentially novel target for antibiotic development. In addition, the catalytic activity of sortase enzymes is utilized in sortase-mediated ligation (SML) engineering approaches for a variety of uses. In SML experiments, engineered variants of S. aureus sortase A (saSrtA) are the most widely used enzymes. Structural analyses of experimental saSrtA structures revealed that the P94 position interacts directly with Y187 when saSrtA is in its inactive conformation. Interestingly, P94 is mutated in the previously engineered pentamutant (or saSrtA5M), to P94R. We wanted to interrogate single mutations at P94 further to characterize its effect on activity and/or substrate specificity. We created 18 P94X mutations (except cysteine) and tested relative activity for 4 substrate sequences: LPATG, LPETG, LPKTG, and LPSTG. We identified several P94 variants that both outperformed and contained differing specificity as compared to P94R. We tested P94A and P94D saSrtA5M variants and found that, depending on the substrate, these variants could outperform saSrtA5M in activity >3-fold. Finally, we compared saSrtA5M and P94D saSrtA5M in a model sortase-mediated ligation reaction using a LPKTG substrate and saw ∼2-fold greater product formation. We argue that future studies which combine rational design and high throughput approaches, e.g., directed evolution, may result in sortase variants with increased SML potential.
Cox-Tigre et al. (Thu,) studied this question.