Honors Program
Honors Program: Embargoed Theses
First Advisor
Limei Zhang
Second Advisor
Jing Zhang
Date of this Version
5-2025
Document Type
Thesis
Citation
Tatineni, R. 2025. Characterization of Residues in TetX8 for Tetracycline Inactivation. Undergraduate Honors Thesis. University of Nebraska-Lincoln.
Abstract
Tetracyclines are a class of broad-spectrum antibiotics historically used to treat bacterial infection via inhibiting bacterial protein synthesis. However, a family of flavin-dependent monooxygenases (FMO) called tetracycline destructases (TetXs) have evolved in bacterial species that confer resistance to many tetracycline substrates. How TetXs specifically recognizes its substrates, and which residues are critical for the enzyme activity remains to be fully understood. This study identified the role of residues in TetX8 for tetracycline inactivation via chimeric mutation compared to its distant homolog LaPhzS, followed by enzymatic activity analysis. LaPhzS is also an FMO that shares a remarkably similar fold to TetX, yet it catalyzes the decarboxylative hydroxylation of phenazine and lacks tetracycline-inactivating properties. In this work, TetX and LaPhzS were used for structural and sequence analysis. TetX8, a TetX family enzyme, was used to generate four TetX8-LaPhzS chimeric mutations to test the essential sequence/structural motifs at the substrate binding pocket of TetX8. Results from the in vivo enzymatic analysis indicate that all four chimeric mutants failed to confer tetracycline resistance. Preliminary purification through Ni-NTA resin suggests that chimera mutants 1-3 significantly affected the protein folding and/or stability, explaining the lack of tetracycline resistance for these mutants. Future steps are needed to produce and isolate correctly folded chimeric mutants to test for tetracycline resistance.
Comments
Copyright Rohan Tatineni 2025.