Design of Ca²⁺‐independent Staphylococcus aureus sortase A mutants

Abstract

The catalytic activity of Staphylococcus aureus sortase A (SaSrtA) is dependent on Ca(2+), because binding of Ca(2+) to Glu residues distal to the active site stabilizes the substrate binding site. To obtain Ca(2+)-independent SaSrtA, we substituted two Glu residues in the Ca(2+)-binding pocket (Glu(105) and Glu(108)). Although single mutations decreased SaSrtA activity, mutations of both Glu(105) and Glu(108) resulted in Ca(2+)-independent activity. Kinetic analysis suggested that the double mutations affect the substrate binding site, without affecting substrate specificity. This approach will allow us to develop SaSrtA variants suitable for various applications, including in vivo site-specific protein modification and labeling.

Site-specific C-terminal and internal loop labeling of proteins using sortase-mediated reactions

“…We have mainly used sortases A derived from Staphylococcus aureus and Streptococcus pyogenes . Versions of Staphylococcus aureus with improved K cat values 32 , as well as mutant versions that do not require Ca ++ ions 33 have been reported and further extend the range of reaction conditions and applications. Streptococcus pyogenes sortase A accepts dialanine (poly)peptides as nucleophiles 34 .…”

“…We commonly use three different sortases: the Ca 2+ dependent sortase A from S. aureus, its mutant version [E105K/E108A 33 ] and sortase A from S. pyogenes; the latter two are Ca 2+ independent. Because sortase is a membrane protein in Gram-positive bacteria, we use versions where the transmembrane domain has been eliminated and replaced with a hexahistidine purification tag.…”

“…The presence of calcium in the reaction buffer precludes the use of phosphate-based buffers. Sortase A from S. pyogenes 34 or a mutant form of S. aureus (E105K/E108A 33 ) are Ca 2+ independent and thus circumvent this limitation.…”

Recent advances in sortase-catalyzed ligation methodology

“…These mutations are localized near the LPXTG-binding groove, likely improving substrate binding. The pentamutant was subsequently enhanced by adding two additional mutations known to eliminate Ca 2+ dependency [12]. Two SrtA staph heptamutants have been described which add either E105K/E108A mutations or E105K/E108Q mutations to the SrtA staph pentamutant backbone [13•, 14, 15•].…”

Site-specific protein modification using immobilized sortase in batch and continuous-flow systems

“…However, both wild type Sortase A and the evolved pentamutant sortase (5M) require Ca 2+ as a cofactor, compromising their utility in applications in the presence of Ca 2+ or in combination with buffers such as phosphate-buffered saline. We recently described a sortase A variant, termed heptamutant sortase A (7M) , which combines the pentamutant version with increased catalytic activity with mutations that render sortase calcium independent 18 , resulting in an enzyme with increased activity and no longer requiring Ca 2+ (Fig. 2) 19 .…”