Date of Completion
2023
Document Type
Honors College Thesis
Department
Biochemistry
Thesis Type
Honors College, College of Arts and Science Honors
First Advisor
Dr. Robert Hondal
Keywords
Biochemistry, Protein Engineering, Singlet Oxygen, Thioredoxin Reductase, Redox, Ergothioneine
Abstract
Ergothioneine is a sulfur containing amino acid found in foods such as mushrooms. It is a unique antioxidant and is a potential vitamin. However, ergothioneine is unable to be incorporated into a peptide or protein because its nitrogen is trimethylated. The amino acid 2- thiohistidine is an analogue of ergothioneine with similar antioxidant properties but can be inserted into a peptide/protein because the nitrogen is bonded to three hydrogens instead of three carbons. The goal of this project is to use protein engineering to replace a catalytic cysteine residue of the C-terminus of Plasmodium falciparum thioredoxin reductase (PfTrxR) with 2- thiohistidine in three variants: PfTrxR–CGGGK2THG, PfTrxR– CG2THKPG2THK, and PfTrxR– CUGGK2THG, where “PfTrxR” represents the body of the enzyme and the letters after the dash are the amino acids of the C-terminal redox center. PfTrxR–CGGGK2THG was hypothesized to gain the ability to reduce free radicals; PfTrxR– CG2THKPG2THK to metabolize singlet oxygen, and the 2TH in PfTrxR–CUGGK2THG to protect the active selenocysteine residue. The catalytic cysteine of PfTrxR is also a sulfur-containing amino acid, but it reduces radicals very poorly. Our data supports the protective effects of 2-thiohistidine on the redox-active residue selenocysteine. It was also observed that the mutant PfTrxR– CUGGK2THG may have gained the ability to metabolize singlet oxygen, instead of PfTrxR– CG2THKPG2THK. This work is an example of protein engineering using a non-standard amino acid in which the mutant gained a new function that was not possible using only the twenty standard amino acids.
Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.
Recommended Citation
Hassett, Elyse C., "Ergothioneine in an Enzyme: Using Protein Engineering to Create Unique Antioxidant Enzymes Containing 2-thiohistidine" (2023). UVM Honors College Senior Theses. 551.
https://scholarworks.uvm.edu/hcoltheses/551