Isopeptide Ligation Catalyzed by Quintessential Sortase A

Abstract

The housekeeping transpeptidase sortase A (SrtA) from Staphyloccocus aureus catalyzes the covalent anchoring of surface proteins to the cell wall by linking the threonyl carboxylate of the LPXTG recognition motif to the amino group of the pentaglycine cross-bridge of the peptidoglycan. SrtA-catalyzed ligation of an LPXTG containing polypeptide with an aminoglycine-terminated moiety occurs efficiently in vitro and has inspired the use of this enzyme as a synthetic tool in biological chemistry. Here we demonstrate the propensity of SrtA to catalyze "isopeptide" ligation. Using model peptide sequences, we show that SrtA can transfer LPXTG peptide substrates to the ⑀-amine of specific Lys residues and form cyclized and/or a gamut of branched oligomers. Our results provide insights about principles governing isopeptide ligation reactions catalyzed by SrtA and suggest that although cyclization is guided by distance relationship between Lys (⑀-amine) and Thr (␣-carboxyl) residues, facile branched oligomerization requires the presence of a stable and long-lived acyl-enzyme intermediate.

Protein Labeling via a Specific Lysine-Isopeptide Bond Using the Pilin Polymerizing Sortase from Corynebacterium diphtheriae

“…Several groups have now optimized this reaction to modify proteins with a range of molecules, including drugs, lipids, sugars, fluorophores, and peptides 13–21 . While Sa SrtA is potent tool, it is almost exclusively used to modify target proteins at their N- or C-termini, while it labels internal lysine side chains as a side reaction with low sequence specificity 15,22,23 . Here we show that a mutationally activated sortase enzyme from Corynebacterium diphtheriae ( Cd SrtA) can site-specifically install a peptide on a protein via a lysine-isopeptide bond.…”

“…Transglutaminases can also modify protein lysine residues, but unlike Cd SrtA 3M , these enzymes exhibit minimal substrate specifity 35,36 . Similarly, Sa SrtA can modify lysines as a side reaction that occurs with minimal specificity and at low efficiency because the lysine ε-amine is not Sa SrtA’s natural substrate 15,22,23 . Chemical methods that modify amino acid side chains have also been developed, but they often require cysteine or non-natural amino acid incorporation into the protein and in some instances harsh reaction conditions 37 .…”

Sortagging: A Robust and Efficient Chemoenzymatic Ligation Strategy

“…Hydrolylsis of the intermediate, another typical side reaction, proceeds always more efficiently 111. 112 However, in the case of the corresponding cyclization even a transient intermediary acyl–enzyme complex can lead to intermolecular isopeptide bonds if the stereochemical positioning of the participating residues is favorable 112…”

Sortase-Mediated Transpeptidation for Site-Specific Modification of Peptides, Glycopeptides, and Proteins

“…31 Roy and co-workers found that SrtA could catalyze an “isopeptide” ligation, namely, transferring peptide substrates with a LPXTG motif to the ε–amino group of Lys residue to form cyclic and/or branched oligomers. 33 Recently, Guo and co-workers 34 applied SrtA to cyclic peptide and glycopeptide synthesis and identified the minimal size required for peptide head to tail cyclization. It was concluded that this method is applicable to the preparation of macrocyclic peptides and glycopeptides containing 15 or more amino acids.…”