Insights into rotaxane formation enable cyclase engineering for lasso peptide diversification

Lasso peptides are a class of ribosomally synthesized and post-translationally modified peptides, which are characterized by their highly stable slip knot-like shape. Lasso peptides are highly diverse in primary sequence and are known to be antibacterial, antiviral, and anticancer, making them a promising scaffold for biomedical applications. A lasso cyclase enzyme uses ATP to fold the linear core peptide into the characteristic lasso peptide shape. These enzymes are known to tolerate different substrates, but their tolerance is limited, and the tolerance rules are poorly defined, which hampers the translational potential of lasso peptides. Understanding the molecular details of how the cyclase contacts the core peptide during folding would enable a deeper understanding of substrate selectivity and facilitate cyclase engineering to access more diverse lasso peptides. However, most lasso cyclase enzymes are not soluble and active when purified, which has limited biochemical characterization of the folding process. The fusilassin pathway is a rare example that can be reconstituted in vitro. I use this system in Chapter 2 to develop a model for lasso peptide folding in the lasso cyclase active site and apply this information to engineer several lasso cyclases with altered substrate tolerances. In Chapter 3, I identify the mycetolassin pathway as another rare example that is functional in vitro and use it to better understand how subtle active site changes contribute to differences in substrate tolerance. These insights answer a long-standing enzymological question about how lasso cyclases interact with their substrates and will facilitate future cyclase engineering campaigns to generate biomedically relevant lasso peptides.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Chemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2024

Author dc:creator

• Barrett, Susanna Elizabeth

Contributors dc:contributor

• Mitchell, Douglas A
• Hergenrother, Paul J
• Nair, Satish K
• Mehta, Angad P

• Lasso Peptide
• Enzyme Mechanism
• Protein Engineering

Statement dc:rights

• Copyright 2024 Susanna Barrett

Language dc:language

eng, en

Handle dc:identifier

https://hdl.handle.net/2142/127453

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F127453