Discipline: Chemistry and Chemical Sciences
Subcategory: Chemistry (not Biochemistry)
Abraham Rotich - Claflin University
Thymidylate is synthesized de novo in all living organisms for replication of genomes. The chemical transformation is reductive methylation of deoxyuridylate at C5 to form deoxythymidylate. All eukaryotes including humans complete this well-understood transformation with thymidylate synthase utilizing (6R)-N5-N10-methylene-5,6,7,8-tetrahydrofolate as both a source of methylene and a reducing hydride. In several human pathogens, thyX-encoded flavin-dependent thymidylate synthase (FDTS) catalyzes the last step in the biosynthesis of thymidylate, one of the four DNA nucleotides. ThyX is absent in humans, rendering FDTS an attractive antibiotic target. In 2002, flavin-dependent thymidylate synthase was discovered as a new pathway for de novo thymidylate synthesis. The goal of this research is to study the mechanism of a Flavin-Dependent Thymidylate Synthase. We hypothesize that analogues of FAD designed to mimic in a flavin dependent thymidylate synthase (FDTS) reaction intermediate can be reconstituted in FDTS. We prepared agar and streaked E. coli containing the gene for FDTS. A bulk culture was then prepared in order to lyse the cells to get the enzyme of interest. A reaction mixture was prepared at room temperature for 15 minutes then used to separate the analogues with the help of HPLC. Trapping and characterization of two consecutive intermediates N5CH3 FAD and C2-CH3 FAD, which together with previous crystal structures indicate that the enzyme?s reduced flavin relays a methylene from the folate carrier to the nucleotide acceptor. These findings indicate a mechanism that is very different from thymidylate biosynthesis in humans, underscoring the promise of FDTS as an antibiotic target to target many human pathogens with low host toxicity.Not Submitted
Funder Acknowledgement(s): Supported by Grants from the Carver College of Medicine, Office of Cultural Affairs and Diversity Initiatives and Claflin University.
Faculty Advisor: Bijoy Dey, firstname.lastname@example.org
Role: I participated in conducting the whole experiment with the guidance of my mentor. I also participated in analyzing results and proposing what we should do in the future.