Isabel Meléndez Morales

Isabel Meléndez Morales

Biography

Isabel Meléndez Morales studies Chemistry at the University of Puerto Rico at Mayagüez. She is especially fascinated by the mechanisms behind enzyme reactions and how these complex processes unfold at a molecular level. In the summer of 2023, Isabel worked on a summer research experience focused on modeling the activity of a synthetic enzyme in a physiological environment using magnesium as a cofactor. This computational project not only sharpened her technical skills, but also deepened her confidence as a scientist. She presented her findings at her first conference, the Annual Biomedical Research Conference for Minoritized Scientists (ABRCMS), where she received an award in the Computational and Systems Biology category for her poster presentation. This experience was more than just a recognition; it was a powerful affirmation of her place in the research world. Beyond academics, Isabel is deeply committed to service. She has worked asa Clinical Research Assistant on studies supporting vulnerable communities and spent three years volunteering at her university’s food bank. She loves exploring the outdoors, playing the piano, and getting lost in a good book. Looking ahead, she hopes to pursue a Ph.D. and contribute to uncovering the molecular stories behind enzyme reactions

Abstract

Interrogating the Re-reduction Mechanism of the active site of Class Ia RNR Through Crystallography

Class Ia ribonucleotide reductase (RNR) is the sole enzyme responsible for catalyzing the de novo synthesis of deoxynucleotides, which are vital for DNA synthesis and repair. During each catalytic cycle, a 2′-hydroxyl group is removed from ribonucleotide to convert it to deoxyribonucleotide. This process requires reduction equivalents, which are provided by two active site cysteines, which then form a disulfide bond. This disulfide bond is re-reduced by a pair of cysteines in the C-terminal tail of the alpha subunit through disulfide exchange. The tail exits in an oxidized form and is then reduced by thioredoxin. We aim to capture structures of RNR in the process of active site reduction by the C-terminal tail using X-ray crystallography. For this, we co-crystallized the alpha subunit with no C-terminal tail (truncated alpha) with a peptide mimicking the C-terminal tail of alpha. I purified the truncated alpha using immobilized metal affinity chromatography, followed by size exclusion chromatography. I used the purified protein to optimize crystallization conditions, obtaining crystals up to 500 μm. Currently, I’m evaluating the diffraction of these crystals with an in-house X-ray source. A
structure depicting the re-reduction process will greatly enhance our understanding of the enigmatic step in deoxyribonucleotide production.