Senior Project Advisor
Jeanine Amacher
Document Type
Project - Campus-only Access
Publication Date
Spring 2025
Keywords
biochemistry, Sortase A, enzyme, FRET fluorescence, activity, interactions, substrate, and endogenous sequence models
Abstract
In Gram-positive bacteria, the Sortase A enzyme (SrtA) catalyzes the ligation of other anchored proteins to the cell surface. The cysteine transpeptidase’s ability to recognize an LPXTG cell wall sorting signal along the substrate and cleave has led to the development of several biochemical applications for the enzyme. Yet, our understanding of the influence of substrate amino acids beyond the LPXTG pentapeptide motif is limited. By understanding how substrate sequence impacts activity, we can expand the versatility of current and future SrtA methods. We applied two models to explore positions along the N-terminus of the cell wall sorting signal (CWSS). The first model was an extension of the LPXTG motif, XLPATG (X = I, P, S, A, D, N, M, G, R, and W). The XLPATG sequences were tested with four SrtA variants for impacts on enzyme activity. The second model sequence was KRQLPSTGE, a Streptococcus pyogenes SrtA (spySrtA) substrate sequence, which was tested with spySrtA. Initial FRET kinetic assays of XLPATG found that Streptococcus agalactiae and Listeria monocytogenes SrtA had little flexibility beyond LPXTG. Yet, Staphylococcus aureus and Streptococcus pyogenes SrtA demonstrated that amino acids in the X position (P5) influenced activity. The KRQLPSTGE model was applied to both FRET assays and Alpha Fold 3 (AF3) modeling and supported the results of the XLPATG assays. Additionally, biochemical and structural analysis supported potential interactions between spySrtA and R/Q substrate residues. This data provides substantial proof that the current CWSS LPXTG model doesn’t fully account for all influential amino acids within Sortase substrates.
Department
Chemistry
Recommended Citation
Andaluz, Devin Janhaskani, "Sortase A: Extended and Endogenous Binding Models" (2025). WWU Honors College Senior Projects. 1011.
https://cedar.wwu.edu/wwu_honors/1011
Type
Text
Rights
Copying of this document in whole or in part is allowable only for scholarly purposes. It is understood, however, that any copying or publication of this document for commercial purposes, or for financial gain, shall not be allowed without the author’s written permission.
Language
English
Format
application/pdf