Designed Streptococcus pyogenes Sortase A Accepts Branched Amines as Nucleophiles in Sortagging

Abstract

Sortase-mediated ligation (sortagging) is commonly performed using the Staphylococcus aureus sortase A (SaSrtA) that strictly recognizes the N-terminal glycine residue. In this work, a rational design of Streptococcus pyogenes sortase A (SpSrtA) for improved transpeptidase activity toward different N-terminal amino acid residues was conducted. The generated variant SpSrtA M3 (E189H/V206I/E215A) showed up to 6.6-fold (vs SpSrtA wild-type) enhanced catalytic efficiency. Additionally, M3 retains the specificity toward N-terminal alanine, glycine, serine residues, as well as branched (at α-carbon) primary amines as wild-type parent. Furthermore, M3 was applied for head-to-tail backbone cyclization of proteins.

Sequence variation in the β7–β8 loop of bacterial class A sortase enzymes alters substrate selectivity

“…Overall, our data are consistent with recent work where a triple mutant of S. pyogenes SrtA (E189H/V206I/E215A, where E215A is a mutation at the β7–β8 −1 position) resulted in 6.6-fold enhanced catalytic efficiency ( 32 ). In addition, K196T in the catalytically enhanced pentamutant saSrtA protein is also located at the β7–β8 −1 position ( 8 ).…”

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

“…6E, Table S1 ). This is consistent with recent work where a triple mutant of S. pyogenes SrtA (E189H/V206I/E215A, where E215A is a mutation at the equivalent β7-β8 −1 position) resulted in a 6.6-fold enhanced catalytic efficiency (26). Notably, K196T in the catalytically enhanced pentamutant saSrtA protein is also located at the β7-β8 −1 position (8).…”

“…Although target sequence recognition by S. aureus SrtA is rigidly selective for a P1’ glycine, this is not true of all Class A sortases, such as those from Streptococcus pneumoniae and Streptococcus pyogenes (10, 30, 34, 35). Building from our previous work, in which spSrtA was found to accept peptides containing Gly-, Ala-, Ser- and other residues at P1’, we have shown here that this broadened substrate scope can be attributed to the sequence of the β7-β8 loop (26). Moreover, variations in β7-β8 loop sequences can substantially impact overall enzyme activity, affording chimeric sortases that outperform their wild-type counterpart i n vitro .…”

“…Given that the β6-β7 loop has been shown to be intimately involved in sortase substrate recognition, we were intrigued that PCA revealed similar levels of variability in the β4-β5 and β7-β8 loops (15). In the case of β7-β8, we were also motivated by previously reported mutations in the β7-β8 loop of saSrtA that have been shown to dramatically modulate sortase reaction rates (8, 17, 26). Therefore, we sought to further explore how the β7-β8 loop affects the activity and substrate specificity of a sortase with narrow substrate tolerance (saSrtA) versus one that is more promiscuous (spSrtA).…”

Challenges in the use of sortase and other peptide ligases for site-specific protein modification

“…Zou et al 66 have recently reported the design of SpSrtA variants with improved transpeptidase activity towards different N-terminal amino acid residues. Based on sequence alignment of sortase A from different species they identified conserved residues near the active site suitable for mutation.…”