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Date of Award

Fall 2025

Document Type

Masters Thesis

Department or Program Affiliation

Chemistry

Degree Name

Master of Science (MS)

Department

Chemistry

First Advisor

Amacher, Jeanine

Second Advisor

Antos, John M.

Third Advisor

Cullati, Sierra

Abstract

Sortases are cysteine transpeptidases which are responsible for the ligation of surface-displayed proteins to the cell wall in gram-positive bacteria. The sortase superfamily is grouped into 6 classes, A–F, with each class recognizing distinct substrates essential for adhesion to host cells, nutrient acquisition, sporulation, and biofilm formation. Apart from being important enzymes to study for understanding bacterial virulence, sortases are also useful for catalyzing in vitro transpeptidation reactions, for example, the synthesis of antibody-drug conjugates. Despite their diversity, however, class A sortases remain the most studied and widely used for protein engineering applications due to higher in vitro activity and a more comprehensive understanding of substrate specificity compared to classes B–F. Here, we investigate the activity of sortase B from Bacillus anthracis (baSrtB) and its endogenous substrate, iron-regulated surface determinant protein C (IsdC). By mass spectrometry, we found that sortase-mediated ligation rates with IsdC were roughly 3-fold greater than ligations using non-native substrate, suggesting that the baSrtB–IsdC complex is driven by interactions outside the sortase recognition motif. Molecular dynamics simulations of this system identified a potential interaction with the IsdC NEAr Transporter (NEAT) domain, which may play a role in enzyme-substrate binding. Sortases A and D from Bacillus anthracis as well as sortase B from Staphylococcus aureus were also studied in silico to probe differences in substrate binding between sortase classes. Finally, we discovered that baSrtB recognizes and ligates substrates to its own N-terminus, which we predict is partially responsible for the relatively low ligation rates observed. Therefore, different protein modification techniques were employed to prevent self-recognition at the N-terminus and boost product formation rates. With this research, we aim to highlight important binding interactions outside of the sorting signal to better inform substrate design and lend insights into working with class B sortases.

Type

Text

Keywords

sortases, cysteine transpeptidase, enzymes, protein engineering, sortase-mediated ligation, structural biology, molecular dynamics, mass spectrometry

Publisher

Western Washington University

OCLC Number

1531273434

Subject – LCSH

Bacillus anthracis--Analysis; Staphylococcus aureus--Analysis; Bacterial proteins--Analysis; Enzymes--Structure; Enzymes--Analysis; Protein engineering--Research

Format

application/pdf

Genre/Form

masters theses

Language

English

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.

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