Semester

Fall

Date of Graduation

2020

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Biochemistry

Committee Chair

Roberta Leonardi

Committee Member

David Smith

Committee Member

Bradley Webb

Committee Member

Jianhai Du

Committee Member

John Hollander

Abstract

Coenzyme A (CoA) is a vital cofactor that is required for a variety of metabolic reactions including the TCA cycle and the synthesis and oxidation of fatty acids, amino acids and ketone bodies. The importance of CoA is underscored by its tight regulation, as prolonged elevations or inability to synthesize adequate amounts of this cofactor lead to severe metabolic dysfunction. Regulation of CoA biosynthesis has been extensively characterized, however less is known about regulation of CoA and its thioesters via degradation. Presently, two CoA-degrading enzymes, Nudt7 and Nudt19 have been identified as regulators of the peroxisomal pool of (acyl-)CoA in the liver and kidneys, respectively. In addition to the peroxisomes, pools of CoA and (acyl-)CoAs exist in the mitochondria and cytosol. Presently, no CoA-degrading enzymes localizing to these compartments have been identified. Nudt8 has been annotated as CoA-degrading enzyme due to its similarity to Nudt7. The CoA-binding motif and Nudix box are highly conserved between Nudt8 and Nudt7; however, the ability of Nudt8 to degrade (acyl-)CoAs had not been established experimentally. We were able to successfully express and purify recombinant Nudt8 and, using enzymatic assays, we confirmed that this enzyme is a CoA diphosphohydrolase specific for free CoA and medium chain acyl-CoAs. Furthermore, we were able to show that Nudt8 localizes to the mitochondrial matrix, positioning it to regulate the largest pool of (acyl-)CoAs in the cell. This research identified a novel CoA-degrading enzyme as a novel means through which mitochondrial CoA, and thus mitochondrial metabolism, is regulated.

Embargo Reason

Publication Pending

Available for download on Saturday, December 04, 2021

Included in

Biochemistry Commons

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