Electronic Thesis and Dissertation Repository

Thesis Format

Monograph

Degree

Master of Science

Program

Biochemistry

Supervisor

Garnham, Christopher

Affiliation

Agriculture and Agri-Food Canada

2nd Supervisor

Shilton, Brian

Co-Supervisor

Abstract

Fumonisins are toxic secondary metabolites produced by pathogenic fungi that infect crops. Ingestion of fumonisin contaminated feed can significantly decrease the health of livestock. Fumonisins are reduced linear polyketides that contain a primary amine and act as competitive inhibitors of ceramide synthase. Novel mechanisms to mitigate fumonisin toxicity are needed. The Garnham and Sumarah labs identified a novel enzyme from Aspergillus niger capable of detoxifying fumonisins by replacing the primary amine with a ketone. In my thesis, I biochemically and biophysically characterized a recombinant version of this fumonisin amine oxidase, termed AnFAO. By solving AnFAO’s X-ray crystal structure and developing spectrophotometric and mass spectrometry assays, I was able to better understand how AnFAO functions at the molecular level to deaminate fumonisins. AnFAO’s characterization allowed for identification of variants with enhanced activity towards fumonisins. These studies will inform how best to deploy AnFAO for remediation of fumonisin contaminated food and feed.

Summary for Lay Audience

Mycotoxins are toxic secondary metabolites produced by pathogenic fungi to enhance survival in their native environment. Fumonisins are a prominent group of these mycotoxins produced by fungi that primarily contaminate corn and corn-based products. Fumonisin contamination of crops is a worldwide problem with significant health and economic impacts. Levels of these toxins in agricultural products are tightly regulated as consumption of fumonisin contaminated food and feed is harmful to humans and livestock. As a result of climate change and global trade, favourable conditions for fungal development are expected to lead to an increase in fumonisin levels. Novel strategies to diminish fumonisin contamination are required to mitigate this threat to farmers, livestock and consumers.

Since the toxic effects of mycotoxins are highly dependent on their structure, specific and efficient modifications by enzymes can be a particularly safe and cost-effective method to remediate contaminated food and feed. The Garnham and Sumarah labs have identified a novel enzyme produced by the fungus Aspergillus niger that is capable of detoxifying fumonisins by replacing the amine functional group with a ketone. I have produced an active recombinant version of this enzyme, termed Aspergillus niger Fumonisin Amine Oxidase (AnFAO).

The goal of my project is to biochemically and biophysically characterize AnFAO to better understand how the enzyme functions at the molecular level. I employed a multi-pronged approach for the characterization of AnFAO and its homologs from various strains of the fungus. First, I utilized spectrophotometric and mass spectrometry assays to confirm AnFAO followed the canonical amine oxidase mechanism. Second, I employed these assays to understand differences in substrate specificity, activity and thermostability between AnFAO homologs. Interestingly, AnFAO homologs were highly thermostable but possessed significant differences in activity towards fumonisins. Finally, I solved AnFAO’s structure via X-ray crystallography to understand how these differences in activity and substrate specificity arise at the molecular level. AnFAO’s structural characterization allowed me to identify variants with enhance fumonisin deamination activity. Altogether this characterization of AnFAO provides a foundation for the future deployment of the enzyme as an efficient, safe, and cost-effective fumonisin remediation tool.

Available for download on Wednesday, June 21, 2023

Included in

Biochemistry Commons

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