Modifying Tryptophan and Indole-Derived Compounds Using Engineered Indole Prenyltransferase Enzymes

<p>Indole Prenyltransferase (IPT) enzymes are present in many microorganisms. They catalyze the transfer of prenyl moieties from natural pyrophosphate donors to tryptophan and other indole-derived small molecules. Prenylation alters the structure of small molecules, enhances their hydrophobicity, and subsequently alters their interaction with cell membranes and receptors. Prenylation of small molecules has been reported to increase cytotoxicity and antimicrobial properties. This suggests that generating enzymes that lead to diprenylation can lead to further improvement in compound properties. PriB is a C-6 IPT that uses dimethylallyl pyrophosphate as a native donor to prenylate tryptophan. PriB has shown broad substrate flexibility, allowing it to modify nonnative donor and acceptor substrates. Structural analysis of PriB active site suggested three key residues that play an important role in the enzyme biocatalytic activity. Thus, site-directed mutagenesis of these three residues was performed in order to expand the active site to facilitate diprenylation, and the encoded enzymes were purified and screened. Our in vitro enzymatic reactions, coupled with HPLC-MS, kinetic data, in addition to 1- and 2D nuclear magnetic resonance spectroscopy, show that the three mutants are capable of catalyzing diprenylation reactions. This work highlights the crucial role of enzyme engineering in biocatalysis, demonstrating its ability to expand enzyme activity across diverse applications.</p>

Name thesis:degree_name

Master of Science (MS)

Level thesis:degree_level

Thesis

Discipline thesis:degree_discipline

Pharmaceutical Sciences

Year dc:date.available

2025

Author dc:creator

• Alexander, Ashley K

Contributors dc:contributor

• Sherif Elshahawi
• Innokentiy Maslennikov
• Cintia Citterio

• Other Pharmacy and Pharmaceutical Sciences

Repository record dc:identifier

https://digitalcommons.chapman.edu/pharmaceutical_sciences_theses/39

OAI identifier oai:identifier

oai:digitalcommons.chapman.edu:pharmaceutical_sciences_theses-1040