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Engineering the specificity of Streptococcus pyogenes sortase A by loop grafting.


ABSTRACT: 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 ?7/?8 loop in determining substrate specificity. We exchanged the ?7/?8 loop in SpSrtA with corresponding ?7/?8 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.

SUBMITTER: Wojcik M 

PROVIDER: S-EPMC7586933 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Engineering the specificity of Streptococcus pyogenes sortase A by loop grafting.

Wójcik Magdalena M   Szala Kamil K   van Merkerk Ronald R   Quax Wim J WJ   Boersma Ykelien L YL  

Proteins 20200621 11


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  ...[more]

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