Sortase A catalyzed reaction pathways: a comparative study with six SrtA variants

Abstract

Sortase A from Staphylococcus aureus (SrtA) has emerged as a useful enzymatic tool to covalently link proteins in a site-directed manner. Despite the proposed specificity of the SrtA-catalyzed reaction for target proteins carrying the amino acid motif LPXTG, enzymatic side reactions are frequently encountered impairing the yield and purity of the transpeptidation product. In this comparative study we investigated reactions of six different variants of SrtA with soluble protein substrates. Besides the transpeptidation reaction generating the desired heterodimeric product, all tested SrtA variants also catalyzed the formation of byproducts (i.e. homooligomeric and hydrolyzed species of the target protein) to different extents. We found that not only the choice of the enzyme, but also the composition and the context of the sorting motif within the particular amino acid sequence of the target protein had a critical impact on the apparent initial rates of the coupling reactions. In addition to the natural sorting signal LPETG, the motif LAETG was also recognized by the investigated SrtA variants. Even though the overall coupling rates with the LAETG-containing target proteins were rather slow, the formation of unwanted byproducts was largely suppressed under these conditions.