The activities to be supported under this proposal center on investigation of the mechanistic aspects of the novel enzymatic chemistry employed by a pair of enzymes that modify ribosomal RNA and consequently are of biological importance as they affect the susceptibility of bacterial ribosomes to inhibition. These enzymes are important members of the radical S-adenosylmethionine (SAM) methyltransferase family. The radical SAM methyltransferases investigated in this study add methyl groups to C2 and C8 carbons of an adenosine nucleotide in the RNA. Unlike the canonical methyl transfer reactions, radical SAM enzymes do not require presence of a nucleophilic atom in the substrate. Rather, they add a methyl group to aromatic amidine carbon atoms, suggesting that these enzymes catalyze methylation by using novel enzymatic chemistry. The preliminary findings indicate that these enzymes have the unique ability to use the same small molecule, S-adenosylmethionine, in two distinct roles: as a precursor of the activating species, and as a co-substrate. To further investigate the unique enzymatic chemistry of Radical SAM methyltransferases, the PI will combine chemical synthesis, RNA modification techniques and mechanistic enzymology to prepare and test substrate analogues that contain nucleotides with altered electronic properties. The importance of individual amino acids within the proteins in enabling catalysis will be investigated through site-directed mutagenesis. Together, these experiments will provide insight into the chemical steps of the methylation mechanism. Broader Impacts. The primary educational objective is to motivate, increase representation, and improve preparedness of students from minority groups, economically disadvantaged backgrounds and first generation college graduates in graduate programs at UCSF. To do so, the PI will participate in outreach efforts to motivate underrepresented undergraduate students to pursue research careers and provide summer research opportunities to this student population to improve their preparedness for graduate school. The outreach activities will be carried out under the umbrella of UCSF's Diversity Committee efforts. The focus will be to raise the awareness about graduate programs and summer undergraduate research opportunities at UCSF among chemistry and biochemistry students at the City College of San Francisco and San Francisco State University, which the PI plans to visit. Additionally, the PI will provide research opportunities for summer undergraduate students in her lab. In summary, as a chemist and mechanistic enzymologist, the PI will attempt to spark interest and provide necessary training in mechanistic, molecular-level understanding of life processes, and improve participation of underrepresented students in research at the interface of chemistry and biology.
