5go5

Publication Abstract from PubMed

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 Staphylococcus aureus cleaves LPXTG-containing proteins at the scissile T-G peptide bond and ligates protein-LPXT to the terminal Gly residue of the nascent cross-bridge of peptidoglycan lipid II precursor. Sortase-mediated ligation ("sortagging") of LPXTG-containing substrates and Gly-terminated nucleophiles occurs in vitro as well as in cellulo in the presence of Ca(2+) and has been applied extensively for protein conjugations. Although the majority of applications emanate from SaSrtA, low catalytic efficiency, LPXTG specificity restriction, and Ca(2+) requirement (particularly for in cellulo 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 Streptomyces avermitilis Biochemical experiments define the attributes of an optimum peptide substrate, demonstrate Ca(2+)-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 LAXTG recognition motif over LPXTG. These results have implications for rational tailoring of substrate tolerance in sortases. Besides, Ca(2+)-independent orthogonal specificity of SavSrtE is likely to expand the sortagging toolkit.

Structure and specificity of a new class of Ca(2+)-independent housekeeping sortase from Streptomyces avermitilis provide insights into its non-canonical substrate preference.,Das S, Pawale VS, Dadireddy V, Singh AK, Ramakumar S, Roy RP J Biol Chem. 2017 Apr 28;292(17):7244-7257. doi: 10.1074/jbc.M117.782037. Epub, 2017 Mar 7. PMID:28270507^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.