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Master of Science (MS)
Antos, John M.
Spiegel, P. Clint
Vyvyan, James R.
Bacterial sortases have been widely studied for their usefulness 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 have made advances towards a substrate bound structure of Streptococcus pneumoniae sortase A (SrtApneu). This enzyme displays a wide tolerance for alternate amino acids within the canonical LPXTG sorting motif. Our strategy involves a non-cleavable peptide 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 isosteres were designed and synthesized, one of which was incorporated into a peptide via solid phase synthesis to produce a non-cleavable sorting signal for SrtApneu. Preliminary analysis of the substrate analog LPAG(keto)G for inhibition of SrtApneu activity in a model transpeptidation reaction suggested that this peptide was an effective inhibitor. Work towards understanding the activity of SrtApneu in relation to its oligomeric state was also undertaken, revealing a strong relationship between the extent of oligomerization and relative activity of SrtApneu, where extensive oligomerization resulted in minimally active samples. Purification of SrtApneu samples was optimized to produce pure monomeric samples of the enzyme, which showed improved transpeptidation activity. This work has helped lay the foundation for future efforts in producing a substrate-bound structure of SrtApneu.
Western Washington University
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Banks, Orion, "Towards the Substrate-bound Structure of Streptococcus pneumoniae Sortase A" (2017). WWU Masters Thesis Collection. 609.