Jackson, S. N.; Blount, J. M.; Croney, K. A.; Lee, D. E.; Ibershof, J. W.; Whitham, K. M.; McCarty, J.; Antos, J. M.; Amacher, J.

Sortases are cysteine transpeptidases located on the surface of Gram-positive bacteria. These critical enzymes facilitate the attachment of proteins to the cell wall, and are potential targets for novel antibiotic development, as well as versatile tools in protein engineering applications. Although there are six classes of sortases recognized, class A sortases (SrtA) are the most widely studied and utilized. SrtA enzymes recognize the canonical Cell Wall Sorting Signal (CWSS), LPXTG, where X=any amino acid, although work in recent years identified additional promiscuity in multiple positions of this recognition motif. Much less is known about Class B sortases (SrtB), which target a distinct sequence, typically with an N terminal Asn, e.g., variations of NPXTG or NPQTN. Although understudied overall, two SrtB enzymes have previously been shown to be specific for heme transporter proteins, and in vitro experiments with the catalytic domains of these enzymes reveal activities significantly worse than SrtA from the same organisms. Here, we use protein biochemistry, structural analyses, and computational simulations to better understand and characterize these enzymes, specifically investigating Bacillus anthracis SrtB (baSrtB) as a model SrtB protein. Structural modeling predicts a plausible enzyme substrate complex, which is verified by mutagenesis of binding cleft residues at several positions. Furthermore, residues N and C-terminal to the pentapeptide recognition motif are critical for observed activity. We also use chimeric proteins to identify a single site that improves baSrtB activity by ~4-fold and use purified protein substrates to validate sortase mediated ligation of two proteins using SrtB enzymes for the first time. Taken together, these studies provide insight into SrtB-target binding as well as evidence that SrtB enzymes can be modified to be of potential use in protein engineering.