Expanding the Scope of Sortase‐Mediated Ligations by Using Sortase Homologues

Abstract

Sortase-catalyzed transacylation reactions are widely used for the construction of non-natural protein derivatives. However, the most commonly used enzyme for these strategies (sortase A from Staphylococcus aureus) is limited by its narrow substrate scope. To expand the range of substrates compatible with sortase-mediated reactions, we characterized the in vitro substrate preferences of eight sortase A homologues. From these studies, we identified sortase A enzymes that recognize multiple substrates that are unreactive toward sortase A from S. aureus. We further exploited the ability of sortase A from Streptococcus pneumoniae to recognize an LPATS substrate to perform a site-specific modification of the N-terminal serine residue in the naturally occurring antimicrobial peptide DCD-1L. Finally, we unexpectedly observed that certain substrates (LPATXG, X=Nle, Leu, Phe, Tyr) were susceptible to transacylation at alternative sites within the substrate motif, and sortase A from S. pneumoniae was capable of forming oligomers. Overall, this work provides a foundation for the further development of sortase enzymes for use in protein modification.

A second specificity-determining loop in Class A sortases: Biochemical characterization of natural sequence variation in chimeric SrtA enzymes

“…In the case of LPATL, this alternate cleavage product was actually the major species obtained following reaction with spSrtAfaecalis. We note here that this capacity for alternate cleavage has been reported previously for wild-type spSrtA, and thus appears to be maintained in the spSrtAfaecalis chimera (27).…”

“…Listeria monocytogenes (spSrtAmonocytogenes), Streptococcus oralis (spSrtAoralis), and Streptococcus suis (spSrtAsuis) (Fig. 4A) (27). To avoid confusion in the numbering of loops with variable lengths, we will hereafter refer to the N-terminal positions of each b7-b8 loop by numbering with respect to the catalytic Cys (b7-b8 +1 , b7-b8 +2 , etc.)…”

Engineering the specificity of Streptococcus pyogenes sortase A by loop grafting

“…Unlike SaSrtA WT, the SpSrtA WT can recognize not only the canonical LPXTG (X being any amino acid) pentapeptide motif but also LPXTA and LPKLG motifs 10 . This allows a somewhat broader scope of substrates for sortase‐mediated ligation 11 . The structure of SpSrtA 9 exhibits some notable differences around the active site, which distinguish it from SaSrtA WT: (a) no Ca 2+ binding site for allosteric activation, (b) a channel that leads to the active site of the enzyme, and (c) an opened β7/β8 loop, creating a prolonged groove 8 .…”

Recent progress in enzymatic protein labelling techniques and their applications

“…101 Several enzyme variants, including an evolved penta-mutant with enhanced catalytic efficiency 102 and a Ca 2+ -independent mutant 103 as well as SrtA homologs from other bacterial sources with different recognition sequences have been reported to meet the requirements of various applications. 104–107 Some of the early uses include protein lipid modification, 108 cyclization, 104 and cell-surface labelling, 109 most of which have been summarized in several reviews. 18,110,111 As an active area of research, an enormous range of applications have been reported since 2013, including but not limited to the semisynthesis of proteins with post-translational modifications, 3,112 protein immobilization on solid surfaces, 113–118 protein labelling on liposomes, 119,120 virus-like particles 121,122 and hydrogels 123–126 as well as cell surface labelling 127 and in vivo protein labelling.…”