Engineered Sortases in Peptide and Protein Chemistry

Abstract

The transpeptidase sortase A of Staphylococcus aureus (Sa-SrtA) is a valuable tool in protein chemistry. The native enzyme anchors surface proteins containing a highly conserved LPxTG sorting motif to a terminal glycine residue of the peptidoglycan layer in Gram-positive bacteria. This reaction is exploited for sortase-mediated ligation (SML), allowing the site-specific linkage of synthetic peptides and recombinant proteins by a native peptide bond. However, the moderate catalytic efficiency and specificity of Sa-SrtA fueled the development of new biocata-lysts for SML, including the screening of sortase A variants form microorganisms other than S. aureus and the directed protein evolution of the Sa-SrtA enzyme itself. Novel display platforms and screening formats were developed to isolate sortases with altered properties from mutant libraries. This yielded sortases with strongly enhanced catalytic activity and enzymes recognizing new sorting motifs as substrates. This minireview focuses on recent advances in the field of directed sortase evolution and applications of these tailor-made enzymes in biochemistry.

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

“…Sortase A from Staphylococcus aureus (saSrtA) was the first of these enzymes discovered and continues to see widespread use for in vitro SML experiments (1,7). Notable improvements in SML technology has occurred in recent years, including strategies for limiting the reversibility of the ligation reaction, and the development of saSrtA variants with dramatically improved catalytic J o u r n a l P r e -p r o o f efficiency (3,8,9). However, as a consequence of the narrow substrate selectivity of saSrtA (10), the majority of SML examples rely on the combination of one ligation partner displaying an LPXTG motif near its C-terminus with another possessing one or more N-terminal glycines.…”

“…However, as a consequence of the narrow substrate selectivity of saSrtA ( 10 ), the majority of SML examples rely on the combination of one ligation partner displaying an LPXTG motif near its C-terminus with another possessing one or more N-terminal glycines. This restricted substrate scope can be advantageous, for example, in the use of SML for labeling specific polypeptides in complex mixtures, but it also represents a limitation for certain applications ( 9 , 11 , 12 ). Highlighting this point, an increasing number of studies have demonstrated that the use of naturally occurring sortases or engineered sortases with altered substrate selectivity offers distinct advantages such as reducing the necessity for point mutations in protein semisynthesis applications ( 12 ), enabling the labeling of endogenous proteins that do not naturally contain the LPXTG motif ( 11 , 13 ), and allowing labeling of multiple sites within the same protein target ( 11 , 14 ).…”

Enzymatic Methods for the Site-Specific Radiolabeling of Targeting Proteins

“…A limit of the SrtA reaction over the hydrolysis of the product is also the low turnover rate of the reaction that necessitates a long incubation time and high enzyme concentration. Further engineering of the enzyme has improved the reaction kinetics of StrA, and it has provided an enzyme that does not depend on the presence of calcium ions and that displays a higher stability to temperature and in the presence of organic solvents (for a recent review on StrA engineering see Freund and Schwarzer [99]). An interesting possibility of StrA derivatization is the co-expression in E. coli of StrA and of the target protein fused at the C-terminus to the sortag.…”

Segmental and site-specific isotope labelling strategies for structural analysis of posttranslationally modified proteins

“…113,114 Overall, Sortase A-catalyzed reactions can provide milligram quantities of ligated product and allow the introduction of PTMs, which makes them an attractive tool for chemoenzymatic segmental labelling of proteins. 115,116…”