Leveraging Sortase A Electrostatics for Powerful Transpeptidation Reactions

Abstract Sortase‐mediated transpeptidation is a powerful biochemical reaction to perform protein engineering. In this work, we leverage the unique electrostatic profile of sortase A pentamutant (SrtA‐5M) to improve SrtA‐5M‐mediated transpeptidations by incorporating short, charged peptidic modules into the substrates. Importantly, the reaction proceeds with a minimal excess of nucleophile and is fast and highly efficient in the low micromolar substrate concentration range. Electrostatic assistance eliminates the need for additives or complex substrate engineering strategies, thereby giving it a broad scope. Our findings also provide fundamental insights into the influence of substrate charge on SrtA‐5M activity, paving the way for further optimization of sortase A‐catalyzed transpeptidation reactions.