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 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 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.
Highlights
At the second position of the pentapeptide motif (ALANT) relative to Pro (ALPNT). This may be partly ascribed to the presence of a bulky Tyr-112 as against an equivalent Ala residue (Ala-104) in specificity of classical housekeeping class A sortase (SaSrtA), which is implicated in substrate recognition [38]
Development of novel sortases endowed with enhanced catalytic efficiency and newer specificity is a topical area of intense interest [19]
Current efforts are centered on generating newer variants of SaSrtA employing rational protein engineering [34, 40] and directed evolution strategies [33, 38, 41]
Summary
S. avermitilis genome encodes at least four putative class E sortases. SrtE3 (SAV4333) is a 230-residue protein composed of an N-terminal transmembrane region (residues 12–32) and a catalytic domain (residues 83–214). A closer inspection of the putative sortase protein substrates encoded in the S. avermitilis genome revealed the presence of neutral residues, such as Asn, Ala, or Gly flanking the LAXTG pentapeptide motif. This prompted us to investigate whether the presence of a Lys residue in YALANTGK exerted any deleterious effect on the transpeptidation reaction. Equilibrium yield in the case of YNLPETGA peptide was found to be about 12–14% as compared with about 9% obtained with YALPNTGK suggesting that flanking residues are important in the context of both LAXTG and LPXTG substrates (Fig. 3B). The distance between the SG atom of the modified Cys and the ND1
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