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Date Permissions Signed

7-21-2017

Date of Award

Summer 2017

Document Type

Masters Thesis

Degree Name

Master of Science (MS)

Department

Chemistry

First Advisor

Antos, John M.

Second Advisor

Spiegel, P. Clint

Third Advisor

Vyvyan, James R.

Abstract

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.

Type

Text

DOI

https://doi.org/10.25710/rafs-5125

Publisher

Western Washington University

OCLC Number

1000095834

Subject – LCSH

Streptococcus pneumoniae--Research; Enzymes--Synthesis--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 thesis for commercial purposes, or for financial gain, shall not be allowed without the author's written permission.

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