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Date of Award
Department or Program Affiliation
Master of Science (MS)
Antos, John M.
Sortases, consisting of classes A-F, are cysteine transpeptidases found in the cell wall of Gram-positive bacteria. They play a crucial role in ligating proteins to the cell wall that are responsible for cell adhesion, immune evasion, host cell invasion, and nutrient acquisition through a transpeptidation reaction. Consequently, they are an attractive therapeutic target. Class A sortases are also utilized in protein engineering applications such as sortase-mediated ligations and sortagging. Despite extensive research in the past two decades, gaps persist in understanding how class A sortases recognize their substrates, primarily due to a lack of structural information on sortases non-covalently bound to substrates. Our work in Chapter 1 presents the first crystal structures of a catalytically inactive Streptococcus pyogenes sortase A (spySrtA) bound to peptide substrates LPATA, LPATS, and a mimetic of the ligation product LPAT-Lipid II. Then, 900 ns molecular dynamic simulations were performed to explore ligand binding dynamics. Additionally, our peptide-bound structures were used to model the acyl-enzyme intermediate of spySrtA-LPAT. Chapter 2 builds on our collaborators' discovery in the Antos lab that certain class A sortases participate in an alternative cleavage motif. Our work involved 18-hour endpoint mass spectrometry assays with various point mutations and chimeric enzymes, revealing that an H143A mutation reduced this alternative cleavage while maintaining near wild type activity. These findings provide insights into target recognition and binding, potentially benefiting therapeutic design and enzyme tuning for protein engineering applications.
Sortase, enzyme, catalysis, alternative cleavage, cysteine transpeptidase, mass spectrometry, structure, intermediate, ligation, mechanism
Western Washington University
Subject – LCSH
Enzymes; Catalysis; Scission (Chemistry); Mass spectrometry
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Vogel, Brandon, "Class A Sortases: Structures and Alternative Substrate Binding and Cleavage" (2023). WWU Graduate School Collection. 1227.