Sortase-mediated ligation (sortagging) is commonly performed using the Staphylococcus aureus sortase A (SaSrtA) that strictly recognizes the N-terminal glycine residue. In this work, a rational design of Streptococcus pyogenes sortase A (SpSrtA) for improved transpeptidase activity toward different N-terminal amino acid residues was conducted. The generated variant SpSrtA M3 (E189H/V206I/E215A) showed up to 6.6-fold (vs SpSrtA wild-type) enhanced catalytic efficiency. Additionally, M3 retains the specificity toward N-terminal alanine, glycine, serine residues, as well as branched (at α-carbon) primary amines as wild-type parent. Furthermore, M3 was applied for head-to-tail backbone cyclization of proteins.