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

Abstract

Gram-positive bacteria contain sortase enzymes on their cell surfaces that catalyze transpeptidation reactions critical for proper cellular function. In vitro, sortases are used in sortase-mediated ligation (SML) reactions for a variety of protein engineering applications. Historically, sortase A from Staphylococcus aureus (saSrtA) has been the enzyme of choice for SML reactions. However, the stringent specificity of saSrtA for the sequence motif LPXTG limits its uses. Here, we use principal component analysis to identify a structurally conserved loop with a high degree of variability in all classes of sortases. We investigate the contribution of this b7-b8 loop, located between the catalytic cysteine and arginine residues and immediately adjacent to the target binding cleft, by designing and testing chimeric sortase enzymes.