The anchoring of the surface proteins to the cell wall in gram-positive bacteria involves a peptide ligation reaction catalyzed by transpeptidase sortase. Most bacterial genomes encode multiple sortases with dedicated functions. Streptococcus pneumoniae (Sp) carries four sortases; a housekeeping sortase (SrtA), and three pilin specific sortases (SrtC1, C2, C3) dedicated to the biosynthesis of covalent pilus. Interestingly, SrtA, meant for performing housekeeping roles, is also implicated in pilus assembly of Sp. The allegiance of SpSrtA to the pathogenic pilus assembly makes it an ideal target for clinical inhibitor development. In this paper, we describe biochemical characterization, crystal structure and peptide substrate preference of SpSrtA. Transpeptidation reaction with a variety of substrates revealed that the enzyme preferred elongated LPXTG sequences and transferred them equally well to both Ala- and Gly-terminated peptides. Curiously, the crystal structure of both wild type and an active site (Cys to Ala) mutant of SpSrtA displayed inter-twined 3D-swapped dimers in which each protomer generated a classic eight-stranded beta-barrel ‘sortase fold'. Size-exclusion chromatography and sedimentation equilibrium measurements revealed the predominant presence of a dimer in equilibrium with its monomer. The crystal structure-based Cys–Cys distance mapping with defined chemical cross-linkers established the existence of 3D-swapped structure in solution. The swapping in SpSrtA, unprecedented for sortase family, may be physiologically relevant and meant to perform regulatory functions.

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