Figure 1.

Truncated sortase enzymes, SrtA_ΔN and SrtA_ΔN59, cleave LPXTG peptides and are activated by calcium ions. (A) Primary structure of wild-type sortase (SrtA), SrtA_ΔN (1) and SrtA_ΔN59 (2) and the position of the conserved leucine (L97), histidine (H120), and cysteine (C184) residues. The N-terminal membrane anchor of S. aureus SrtA is indicated as a black box. (B) Coomassie blue-stained SDS/PAGE displays the migration of purified SrtA_ΔN (lane 1) and SrtA_ΔN59 (lane 2). (C) SrtA_ΔN59 and SrtA_ΔN were incubated with d-LPETG-e peptide, and cleavage was measured as an increase in fluorescence. Addition of 2 mM Ca²⁺ or Mn²⁺ ions to the reaction buffer stimulated enzyme activity 8-fold, whereas K⁺ had no effect on cleavage. (− marks control.)