Date of Award

Spring 5-2025

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Pharmaceutical Sciences

First Advisor

Sherif Elshahawi

Second Advisor

Innokentiy Maslennikov

Third Advisor

Cintia Citterio

Abstract

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.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

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