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Engineering the specificity of Streptococcus pyogenes sortase A by loop grafting
Author(s) -
Wójcik Magdalena,
Szala Kamil,
Merkerk Ronald,
Quax Wim J.,
Boersma Ykelien L.
Publication year - 2020
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.25958
Subject(s) - sortase a , peptidoglycan , pentapeptide repeat , sortase , streptococcus pyogenes , mutant , virulence , microbiology and biotechnology , bacillus anthracis , staphylococcus aureus , biochemistry , biology , chemistry , enzyme , bacteria , peptide , genetics , gene
Sortases are a group of enzymes displayed on the cell‐wall of Gram‐positive bacteria. They are responsible for the attachment of virulence factors onto the peptidoglycan in a transpeptidation reaction through recognition of a pentapeptide substrate. Most housekeeping sortases recognize one specific pentapeptide motif; however, Streptococcus pyogenes sortase A (SpSrtA WT) recognizes LPETG, LPETA and LPKLG motifs. Here, we examined SpSrtA's flexible substrate specificity by investigating the role of the β7/β8 loop in determining substrate specificity. We exchanged the β7/β8 loop in SpSrtA with corresponding β7/β8 loops from Staphylococcus aureus (SaSrtA WT) and Bacillus anthracis (BaSrtA WT) . While the BaSrtA‐derived variant showed no enzymatic activity toward either LPETG or LPETA substrates, the activity of the SaSrtA‐derived mutant toward the LPETA substrate was completely abolished. Instead, the mutant had an improved activity toward LPETG, the preferred substrate of SaSrtA WT.