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.
","abstract_html":"<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>","abstract_has_math":false,"creators":["Alexander, Ashley K"],"institution":null,"degree_name":"Master of Science (MS)","degree_level":"Thesis","degree_discipline":"Pharmaceutical Sciences","degree_department":null,"school":null,"contributors":["Sherif Elshahawi","Innokentiy Maslennikov","Cintia Citterio"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2025,"date_issued":"2025-05-01T07:00:00Z","date_published":"2025-05-01T07:00:00Z","updated_at":"2026-07-24T01:38:43Z","subjects":["Other Pharmacy and Pharmaceutical Sciences"],"languages":[],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://digitalcommons.chapman.edu/pharmaceutical_sciences_theses/39","outbound_label":"Repository record","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Sherif Elshahawi","Innokentiy Maslennikov","Cintia Citterio"]},{"key":"dc:creator","label":"Author","values":["Alexander, Ashley K"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.available","label":"Dc Date Available","values":["2027-05-08T07:00:00Z"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Pharmaceutical Sciences"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Thesis"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Master of Science (MS)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Other Pharmacy and Pharmaceutical Sciences"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://digitalcommons.chapman.edu/pharmaceutical_sciences_theses/39"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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.
"]},{"key":"dc:source","label":"Dc Source","values":["Alexander, A K. Modifying Tryptophan and Indole-Derived Compounds Using Engineered Indole Prenyltransferase Enzymes. [master’s thesis]. Irvine, CA: Chapman University; 2025. https://doi.org/10.36837/chapman.000682"]},{"key":"dc:title","label":"Title","values":["Modifying Tryptophan and Indole-Derived Compounds Using Engineered Indole Prenyltransferase Enzymes"]}]}],"canonical_facts":{"dc:contributor":["Sherif Elshahawi","Innokentiy Maslennikov","Cintia Citterio"],"dc:creator":["Alexander, Ashley K"],"dc:date.available":["2027-05-08T07:00:00Z"],"dc:description.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.
"],"dc:identifier":["https://digitalcommons.chapman.edu/pharmaceutical_sciences_theses/39"],"dc:source":["Alexander, A K. Modifying Tryptophan and Indole-Derived Compounds Using Engineered Indole Prenyltransferase Enzymes. [master’s thesis]. Irvine, CA: Chapman University; 2025. https://doi.org/10.36837/chapman.000682"],"dc:subject":["Other Pharmacy and Pharmaceutical Sciences"],"dc:title":["Modifying Tryptophan and Indole-Derived Compounds Using Engineered Indole Prenyltransferase Enzymes"],"thesis:degree_discipline":["Pharmaceutical Sciences"],"thesis:degree_level":["Thesis"],"thesis:degree_name":["Master of Science (MS)"]},"updated_at":"2026-07-24T01:38:43Z"}