Structural analysis via X-ray crystallography of an engineered carbon methyltransferase, a phytoxic phosphonate and its modifying enzyme, and a chimeric tyrosinase

Enzymes are the workhorses of the biochemical world. They catalyze numerous reactions under mild conditions. Throughout existence, many enzymes have become specialized to catalyze specific reactions. Many of these reactions are unique and highly desirable for industrial applications; therefore, enzymes are often repurposed from their native environment to serve in these roles. In some cases, the wild-type enzyme functions as intended. However, most cases require retooling of the enzyme to ensure it functions at maximum efficiency in the new environment. In the second chapter, I introduce S-adenosylmethionine (SAM) dependent methyltransferases. These enzymes transfer a -CH3 group from SAM onto a substrate. This alkylation is routinely done via synthetic chemistry but can require extreme conditions and hazardous solvents. Engineering a methyltransferase to accept a variety of substrates can circumvent such difficulties. This chapter describes my structural characterization of the methyltransferase SgvM. My data guided the engineering efforts of collaborators, resulting in a substrate-tolerant variant enzyme. I also describe my characterization of this variant. In the third chapter, I introduce a specialized metabolite called pantaphos. Characterized by a stable carbon-phosphorus bond, pantaphos is a phosphonate. Members of this family of molecules are readily available in the commercial market as components of herbicides, water softeners, and certain medications. Identified by my collaborator as the phytotoxin responsible for Pantoea ananatis derived onion center rot, pantaphos has the potential to be utilized as an herbicide. Here, I describe my structural characterization of pantaphos and one of the enzymes involved in its synthesis. In the fourth chapter, I describe my structural characterization of a chimeric enzyme engineered for specificity. My collaborators achieved this by fusing a promiscuous enzyme to a substrate-binding domain. Such constructs are already widely used for targeted reactions, such as gene editing or labeling. However, most chimeras utilize only a linker to connect the domains. This results in some loss of efficiency as the binding domain may not always be oriented to direct the substrate into the active site. Taking inspiration from the multi-domain proteins involved in the posttranslational processing of ribosomally synthesized peptides (RiPPs), my collaborators used an in silico approach to additionally design an interface between the binding domain of Fyn tyrosine kinase and a tyrosinase.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biochemistry

Grantor

University of Illinois Urbana-Champaign

Year dc:date

2025

Author dc:creator

• Kuzelka, Kaylee Pauline

Contributors dc:contributor

• Nair, Satish K
• van der Donk, Wilfred
• Metcalf, William W
• Stadtmueller, Beth M

• Crystallography
• enzymes
• structural biology
• enzyme engineering

Statement dc:rights

• Copyright 2025 Kaylee Kuzelka

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/129706

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F129706