Analysis of the Enzymatic Capacity of MitoNEET
Board Location: #133
Discipline: Biological Sciences
Subcategory: Biochemistry (not Cell and Molecular Biology and Genetics)
Session: 1
Taylor Bias - Ball State University
Co-Author(s): Mary E. Konkle, Ball State University, Muncie, IN
MitoNEET is a protein that contains a [2Fe-2S] cluster ligated by one histidine and three cysteine residues. This protein is a drug target for human diseases such as Parkinson’s disease, type-two diabetes, and breast cancer, the problem is a lack of fundamental understanding of mitoNEET’s chemical reactivity and how it contributes to disease pathologies. MitoNEET is known to react with thiol containing compounds, which are an emerging priority for understanding oxidative stress in the cell. This study evaluated mitoNEET for enzymatic activity towards the sulfur-containing amino acids cysteine and homocysteine. The outcomes were evaluated using HPLC and GC-MS instrumentation after derivatization of the product(s). MitoNEET enzymatically converts cysteine to cystine and this reaction can be observed through GC-MS following derivatization with N-tert-Butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA). Kinetic analysis indicates the presence of a yet-unknown intermediate. Interestingly, mitoNEET has a different activity profile after modification with the coenzyme pyridoxal 5’-phosphate (PLP) at Lys55. The mitoNEET-PLP enzyme complex converts cysteine and 2-oxoglutarate to glutamate and 3-mercaptopyruvate in a transamination reaction. The starting materials cysteine and 2-oxoglutarate along with the product glutamate can also be observed by GC-MS with MTBSTFA. The product 3-mercaptopyruvate can be derivatized with monobromobimane (MBB) and analyzed by HPLC. Additionally, mitoNEET can also use homocysteine as the amino donor to convert 2-oxoglutarate to glutamate in a transamination. Further analysis interrogates how other molecules that are known ligands of mitoNEET effect the transaminase activity. Taken together, this work will be of broad importance not only to scientists who study mitoNEET, but also to those who study how the metabolism of thiol-containing compounds contribute and respond to oxidative stress.
Funder Acknowledgement(s): NSF-HRD-1618408, Ball State IN LSAMPNSF-1609440, 1806606, Mary Konkle and Michael Menze
Faculty Advisor: Mary E. Konkle, mekonkle@bsu.edu
Role: In this study, I evaluated mitoNEET for enzymatic activity with the guidance of a graduate student. I evaluated these outcomes using HPLC and GC-MS instrumentation after derivatization of the product(s). We found that MitoNEET enzymatically converts cysteine to cystine and that it has a different activity profile after modification with the coenzyme pyridoxal 5’-phosphate (PLP) at Lys55. The mitoNEET-PLP enzyme complex converts cysteine and 2-oxoglutarate to glutamate and 3-mercaptopyruvate in a transamination reaction.