Poster abstracts
Poster number 10 submitted by Aouss Azzouz
Generating ProXp-x Active Site Mutations to Determine the Mechanism of D-aa-tRNA Selection
Aouss Azzouz (Department of Chemistry, Earlham College), Gary Coker (Department of Chemistry, Earlham College), Jalyn Kelsey (Department of Chemistry, Earlham College), Amanda Vasconcellos (Department of Chemistry, Earlham College), Alana Weaver (Department of Chemistry, Earlham College), Alexandra Kuzmishin Nagy (Department of Chemistry, Earlham College)
Abstract:
Aminoacyl-tRNAs that carry D-amino acids, rather than the L-enantiomers used in protein synthesis, can deplete the pool of charged tRNA available to the cell, resulting in impaired bacterial growth and cytotoxicity.[1, 2] Bacteria that are sensitive to the presence of D-amino acids show reduced fitness and growth when cultured in media rich in D-enantiomers.[1, 3, 4] ProXp-x is a trans-editing enzyme found in soil bacteria that can hydrolyze D-amino acids from aminoacyl-tRNAs,[5] however the specific mechanism for D-amino acid editing has not been elucidated. It is also not understood if this mechanism protects the organism from D-amino acids. This research aims to understand how ProXp-x selects and binds mischarged D-aminoacyl-tRNA (D-aa-tRNA) species within the active site. Conserved active site residues of ProXp-x were selected for mutation; previous work implicated candidate residues in catalysis or substrate orientation.[5] Site-directed mutagenesis was used to mutate active site residues of wild type (WT) and K45A ProXp-x encoded on pET15b plasmids. The K45A mutation renders ProXp-x and other INS-like Superfamily members catalytically inactive, allowing for studies of substrate binding without catalysis to degrade the D-aa-tRNA. These amino acid residues are hypothesized to play a direct role in orienting the substrate within the active site, however their specific interactions are unknown. The use of active site point mutations provides a gateway to understanding how these residues are involved in the activity of ProXp-x. Future studies include using these ProXp-x variants in kinetic and binding assays to assess how the targeted residues contribute to substrate selection.
References:
1. Soutourina, O., et al., Formation of D-tyrosyl-tRNATyr accounts for the toxicity of D-tyrosine toward Escherichia coli. J Biol Chem, 2004. 279(41): p. 42560-5.
2. Englander, M.T., et al., The ribosome can discriminate the chirality of amino acids within its peptidyl-transferase center. PNAS, 2015. 112(19): p. 6038-43.
3. Soutourina, J., P. Plateau, and S. Blanquet, Metabolism of D-aminoacyl-tRNAs in Escherichia coli and Saccharomyces cerevisiae cells. J Biol Chem, 2000. 275(42): p. 32535-42.
4. Soutourina, J., S. Blanquet, and P. Plateau, D-tyrosyl-tRNA(Tyr) metabolism in Saccharomyces cerevisiae. J Biol Chem, 2000. 275(16): p. 11626-30.
5. Bacusmo, J.M., et al., Quality control by trans-editing factor prevents global mistranslation of non-protein amino acid alpha-aminobutyrate. RNA Biol, 2017: p. 1-10.
Keywords: aminoacyl-tRNA, trans-editing, D-amino acids