Structure and specificity of a new class of Ca-independent housekeeping sortase from provide insights into its non-canonical substrate preference.

Surface proteins in Gram-positive bacteria are incorporated into the cell wall through a peptide ligation reaction catalyzed by transpeptidase sortase. Six main classes (A-F) of sortase have been identified of which class A sortase is meant for housekeeping functions. The prototypic housekeeping sortase A (SaSrtA) from cleaves LPTG-containing proteins at the scissile T-G peptide bond and ligates protein-LPT to the terminal Gly residue of the nascent cross-bridge of peptidoglycan lipid II precursor. Sortase-mediated ligation ("sortagging") of LPTG-containing substrates and Gly-terminated nucleophiles occurs as well as in the presence of Ca and has been applied extensively for protein conjugations. Although the majority of applications emanate from SaSrtA, low catalytic efficiency, LPTG specificity restriction, and Ca requirement (particularly for applications) remain a drawback. Given that Gram-positive bacteria genomes encode a variety of sortases, natural sortase mining can be a viable complementary approach akin to engineering of wild-type SaSrtA. Here, we describe the structure and specificity of a new class E sortase (SavSrtE) annotated to perform housekeeping roles in Biochemical experiments define the attributes of an optimum peptide substrate, demonstrate Ca-independent activity, and provide insights about contrasting functional characteristics of SavSrtE and SaSrtA. Crystal structure, substrate docking, and mutagenesis experiments have identified a critical residue that dictates the preference for a non-canonical LATG recognition motif over LPTG. These results have implications for rational tailoring of substrate tolerance in sortases. Besides, Ca-independent orthogonal specificity of SavSrtE is likely to expand the sortagging toolkit.