Designed Streptococcus pyogenes Sortase A Accepts Branched Amines as Nucleophiles in Sortagging

18/11/2020
 

Zhi Zou, Maximilian Nöth, Felix Jakob, and Ulrich Schwaneberg. Designed Streptococcus pyogenes Sortase A Accepts Branched Amines as Nucleophiles in Sortagging. Bioconjugate Chemistry, 2020

Sortase-mediated ligation (sortagging) is commonly performed using the Staphylococcus aureus sortase A (SaSrtA) that strictly recognizes the N-terminal glycine residue as sortase nucleophile. In this work, a rational design (site-directed mutagenesis close to the active site and β6/β7 loop engineering) 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 to branched primary amines (branched at the α-carbon) similar to the wild-type parent. Furthermore, M3 was applied for head-to-tail backbone cyclization of proteins.SpSrtA was engineered for increased activity towards differen N-terminal amino acid resiudes (alanine, glycine, and serine) and primary amines (branched at the α-carbon) by rational design. Copyright (2020), American Chemical Society.

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