BRC-BIO: Basis of L- and D-Amino Acid Substrate Recognition by Bacterial Aminoacyl-tRNA Editing Enzyme

This research investigates how bacteria protect themselves from toxic D-amino acids. Proteins perform important functions in cells. Cells build proteins using small molecules, called amino acids. Amino acids occur in two mirror image forms: L-amino acids and D-amino acids. L-amino acids are used in proteins, but D-amino acids are not. It is not understood why L-amino acids are specifically used in proteins, but it is known that using D-amino acids can be fatal to bacterial cells. Enzymes within a cell can typically recognize either L- or D-amino acids. There is an enzyme, ProXp-x, which is an exception; it recognizes both. This project will investigate how the enzyme, ProXp-x, prevents D-amino acid use in proteins of soil-dwelling bacteria. This research will determine how ProXp-x recognizes D- vs. L-amino acids, and how ProXp-x protects bacterial cells from D-amino acid toxicity. The findings will fill gaps in the field’s understanding of protein creation and enzyme function, adding to the field’s fundamental knowledge of enzymes and how cells adapt to stress. This project will also train a team of undergraduate researchers, create several exhibits and games about molecular biology and biochemistry for the Joseph Moore Museum (a natural history museum on Earlham College’s campus), and lead to the publication of classroom materials to use the project as an interactive laboratory experience. The project will characterize how ProXp-x identifies and binds its targets and determine if its activity prevents D-amino acid toxicity in living bacteria. D-amino acids can be mischarged onto tRNAs, preventing accurate protein production. Many bacteria encode D-aminoacyl-tRNA deacylases (DTDs), an enzyme that removes D-amino acids from D-aa-tRNAs. Soil-dwelling Rhizobia bacteria do not encode DTDs, but do encode ProXp-x. Unlike DTDs, ProXp-x does not recognize all D-aa-tRNAs or Gly-tRNA. Instead, ProXp-x activity appears dependent on the specific combination of amino acid and tRNA in the aa-tRNA. The project will employ undergraduates to conduct mentored research to study ProXp-x in Rhodopseudomonas palustris. Project aims include using kinetic assays and binding experiments with purified protein and aa-tRNAs to determine how ProXp-x distinguishes between mischarged L- and D-aa-tRNAs, specifically identifying the tRNA features recognized by ProXp-x and the active site residues that recognize the aminoacyl-moiety. Students will also measure R. palustris growth phenotypes and D-aa-tRNA levels in cells to determine if ProXp-x prevents D-amino acid toxicity. Taken together, the project expands the field’s understanding of protein synthesis, enzyme function, and how bacteria contend with D-amino acids. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.