Mutation-induced heterogeneity of the β7-β8 loop of the Staphylococcus aureus class A sortase leading to enhanced catalytic efficiency characterized by NMR and enzyme kinetics
Bacterial sortase enzymes are cysteine transpeptidases at the surface of Gram-positive bacteria that ligate substrates to the cell wall. In addition, these enzymes are powerful tools in protein engineering applications via sortase-mediated ligation (SML) due to their covalent attachment of two substrates, with one containing a pentapeptide recognition motif with sequence LPXTG, where X=any amino…
Sortase enzymes, which are cysteine transpeptidases on the surface of Gram-positive bacteria, are utilized in protein engineering via sortase-mediated ligation (SML). The class A sortase from Staphylococcus aureus (saSrtA) is the most widely used SML variant. Researchers have found a mutation in the β7-β8 loop of saSrtA, specifically position P94, that enhances catalytic efficiency.
This mutation directly interacts with a structurally conserved loop near the active site of the wild-type saSrtA in its inactive conformation. The mutation affects the enzyme's relative activity and specificity for the P2 position in the LPXTG recognition motif, primarily due to Km effects. The study further investigates the structural changes in the active, apo state of saSrtA when the P94D mutation is introduced, as well as mutations in Y187, a residue hypothesized to interact directly with P94.
Using 1H-15N NMR experiments, the researchers compare spectra between inactive variants of saSrtA with and without the P94D mutation. They also use NMR to calculate relative binding affinities for a pentapeptide substrate to these variants and inactive saSrtA5M. The findings provide atomic details regarding the importance of the P94 residue in saSrtA substrate recognition.
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