Bacterial sortases have been widely studied for their utility in protein modification, however, the variable substrate specificity and activity between homologs of these enzymes is not yet fully characterized. To attempt to further understand sorting signal recognition, we are working towards a substrate bound structure of sortase A from Streptococcus pneumoniae (SrtApneu). This enzyme displays a wide tolerance for alternate amino acids within the canonical LPXTG sorting motif. Our strategy involves a non-cleavable substrate analog that can be docked into the active site, allowing for elucidation of a structure displaying the key contacts that allow the enzyme to recognize alternate sorting signals. To this end, ketomethylene-linked “dipeptide” isosteres were synthesized and incorporated into peptides via solid phase synthesis to produce non-cleavable sorting signals accepted by SrtApneu. These substrate analogs were found to inhibit SrtApneu activity in a model transpeptidation reaction. Additionally, work towards understanding the activity of SrtApneu in relation to its oligomeric state will also be presented.

Substrate analogs for characterizing the substrate tolerance of S. pneumoniae sortase A

253rd National Meeting (2017)

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