Structure and specificity of a new class of Ca2+-independent housekeeping sortase from Streptomyces avermitilis provide insights into its non-canonical substrate preference

Das, Sreetama and Pawale, Vijaykumar S and Dadireddy, Venkatareddy and Singh, Avinash Kumar and Ramakumar, Suryanarayanarao and Roy, Rajendra P (2017) Structure and specificity of a new class of Ca2+-independent housekeeping sortase from Streptomyces avermitilis provide insights into its non-canonical substrate preference. In: JOURNAL OF BIOLOGICAL CHEMISTRY, 292 (17). pp. 7244-7257.

PDF Jou_Bio_Che_292-17_7244_2017.pdf - Published Version Restricted to Registered users only Download (3MB) | Request a copy

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 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 Ca2+ and has been applied extensively for protein conjugations. Although the majority of applications emanate from SaSrtA, low catalytic efficiency, LPXTG specificity restriction, and Ca2+ 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 wildtype 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 Ca2+-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, Ca2+-independent orthogonal specificity of SavSrtE is likely to expand the sortagging toolkit.

Item Type:	Journal Article
Publication:	JOURNAL OF BIOLOGICAL CHEMISTRY
Department/Centre:	Division of Physical & Mathematical Sciences > Physics
Date Deposited:	03 Jun 2017 09:40
Last Modified:	03 Jun 2017 09:40
URI:	http://eprints.iisc.ac.in/id/eprint/57104

Actions (login required)

View Item