Download MendeleyStructure and specificity of a new class of Ca2+-independent housekeeping sortase from Streptomyces avermitilis provide insights into its non-canonical substrate preference.
Das, S., Pawale, V.S., Dadireddy, V., Singh, A.K., Ramakumar, S., Roy, R.P.(2017) J Biol Chem 292: 7244-7257
- PubMed: 28270507
- DOI: https://doi.org/10.1074/jbc.M117.782037
- Primary Citation of Related Structures:
5GO5 - PubMed Abstract:
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 LP X TG-containing proteins at the scissile T-G peptide bond and ligates protein-LP X T to the terminal Gly residue of the nascent cross-bridge of peptidoglycan lipid II precursor. Sortase-mediated ligation ("sortagging") of LP X TG-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, LP X TG 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 LA X TG recognition motif over LP X TG. 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.
Organizational Affiliation:
From the Department of Physics, Indian Institute of Science, Bangalore 560012, and.
