Staphylococcal Enterotoxin M Exhibits Thrombin-like Enzymatic Activity.

To express and purify staphylococcal enterotoxin M (SEM) using immobilized metal affinity chromatography (IMAC), a signal peptide-truncated (ΔNsp) wild-type SEM (SEMWT) was N-terminally fused in pET-28a(+) to a polyhistidine tag (His6×-) and thrombin cleavage site (TCS; LVPR↓GS), generating His6×-TCS-ΔNspSEMWT. Unexpectedly, 4 °C desalting reduced the fusion protein's molecular weight by ~2.0 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). N-terminal sequencing and mass spectrometry identified cleavage specifically at the arginine (R) and glycine (G) peptide bond (R-G bond) within the TCS motif. AlphaFold 3 revealed an exposed serine protease catalytic triad: histidine 172, serine 178, and aspartic acid 212 (H172/S178/D212) in the β-grasp domain, suggesting intrinsic thrombin-like activity (TLA). Sequential IMAC and size-exclusion high-performance liquid chromatography (SE-HPLC) purification eliminated contaminant concerns, while chromogenic substrate S-2238 (S-2238) assays demonstrated increasing specific activity and purification fold, supporting intrinsic TLA. Critically, the mutation of serine at position 178 to alanine (His6×-TCS-ΔNspSEMS178A) abolished TLA but preserved the secondary/tertiary structure, confirming the activity's origin within the wild-type construct. Molecular dynamics (MD) simulations probed the atomistic mechanism for specific R-G bond cleavage. This work establishes a foundation for understanding ΔNspSEMWT's TLA.