Feustel, Kelsey
Kelsey is in the Biochemistry, Molecular and Structural Biology Graduate Program, and joined the CMB Training Program in 2022.
Mentor: Dr. Cathy Clarke
Coenzyme Q (CoQ or ubiquinone) is a redox-active lipid molecule that acts as an electron carrier in the mitochondrial electron transport chain, aiding in the mitochondrial production of ATP. In Saccharomyces cerevisiae, at least 14 nuclear encoded proteins are required for efficient mitochondrial biosynthesis of CoQ6, an isoform of CoQ with a hexaprenyl “tail” of six isoprene units. Many of the Coq polypeptides (Coq3-Coq9 and Coq11) localize to the matrix side of the inner mitochondrial membrane where they assemble into a high molecular mass complex known as the CoQ Synthome. The correct assembly of this complex is required for efficient CoQ biosynthesis, as it is destabilized by individual deletion of COQ genes, resulting in severe defects in CoQ biosynthesis. Recently, the polypeptide Coq11 was identified as a novel member of the CoQ Synthome. Deletion of COQ11 has been shown to significantly reduce, but not abolish, de novo CoQ6 biosynthesis. Despite its impaired CoQ6 biosynthesis, the coq11Δ mutant appears to display a putatively more stable or enlarged CoQ Synthome and retains the antioxidant properties afforded by ubiquinol (CoQH2). While several roles for Coq11 have been hypothesized, its function in CoQ6 biosynthesis has not yet been fully elucidated.
Sequence analyses have identified Coq11 as a member of the short-chain dehydrogenase/reductase (SDR) superfamily, specifically as a member of subgroup five of the atypical SDRs. The SDRs constitute a large family of catalytically diverse enzymes that, despite having low pairwise sequence identities, share a relatively conserved catalytic triad or tetrad, several loosely conserved sequence motifs, and a conserved N-terminal Rossmann-fold, a protein structural motif utilized in the binding of a dinucleotide cofactor.
My research aims to characterize the function of Coq11 using its identity as a member of the SDR superfamily. CRISPR-Cas9 mediated genome editing will be used to introduce point mutations into the genome of S. cerevisiae at residues that comprise the hypothesized Coq11 catalytic tetrad. Strains harboring these point mutations will be phenotypically characterized by examining respiratory capacity, de novo CoQ6 biosynthesis via mass spectrometry, and retention of the antioxidant protection afforded by ubiquinol. Additionally, the effect of these Coq11 point mutations on the assembly and stability of the CoQ Synthome will be assessed using 2D Blue-Native/SDS-PAGE and Western Blot analyses. Overall, the findings of my research will provide insight into the function of an atypical SDR required for efficient CoQ6 biosynthesis in S. cerevisiae.
