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Probing the Evolution of New Specificities in Aminoacyl-tRNA Synthetases

Gilreath, Marla S.
http://rave.ohiolink.edu/etdc/view?acc_num=osu1308241476

Abstract Details

2011, Master of Science, Ohio State University, Biochemistry.
Bacterial elongation factor P (EF-P) is a poorly understood soluble protein that has been shown to enhance the first step of peptide bond formation through an interaction with the ribosome and initiator tRNA. Homologous proteins have been found in both archaeal and eukaryotic systems, known as aIF5A and eIF5A, respectively. eIF5A, which was recently shown to increase translation elongation rates, is post-translationally modified at a highly conserved lysine residue through the addition of the rare amino acid hypusine. A similar pathway was recently elucidated for EF-P, in which EF-P is post-translationally modified by the enzymes PoxA and YjeK at lysine 34, corresponding to a homologous site of hypusination in a/eIF5A. As a paralog of class II LysRS, PoxA catalyzes the addition of lysine onto EF-P, but is incapable of modifying tRNA. YjeK is a 2,3-(β)-lysine aminomutase and is responsible for converting lysine to β-lysine, which PoxA was recently shown to recognize as a preferred substrate for EF-P modification. The amino acid binding pockets of LysRS and PoxA are highly conserved, with the exception of two residues, Gly465 and Ala229 of Geobacillus stearothermophilus LysRS and Ala298 and Ser76 of Salmonella Typhimurium PoxA. Despite their substantial active site similarity, PoxA exhibits a significantly higher KM value for activation of lysine as compared to LysRS. This suggests that the two divergent residues in the active site determine the specificity for substrate recognition and binding, as well as optimal enzymatic activity, of PoxA. To investigate the mechanisms of α- versus β-amino acid recognition in the divergent evolution of LysRS and PoxA, three amino acid replacements were made in the LysRS active site. Kinetic parameters for ATP/PPi exchange reactions were determined for wild type, A233S, and G469A LysRS and aminoacylation reactions were carried out to further characterize the activity of each variant. Results indicate that while A233S behaves like the wild type, G469A and A233S/G469A significantly decrease the ability of LysRS to form stable lysyl-adenylates. A233S was able to shift the substrate specificity of LysRS to recognize (S)-β-lysine, indicating that few active-site substitutions are necessary to facilitate changes in the substrate specificity of an aaRS.
Michael Ibba, PhD (Advisor)
Venkat Gopalan, PhD (Advisor)
47 p.
Biochemistry
Elongation Factor P; PoxA; aminoacyl-tRNA synthetase

Recommended Citations

Citations

  • Gilreath, M. S. (2011). Probing the Evolution of New Specificities in Aminoacyl-tRNA Synthetases [Master's thesis, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1308241476

    APA Style (7th edition)

  • Gilreath, Marla. Probing the Evolution of New Specificities in Aminoacyl-tRNA Synthetases. 2011. Ohio State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1308241476.

    MLA Style (8th edition)

  • Gilreath, Marla. "Probing the Evolution of New Specificities in Aminoacyl-tRNA Synthetases." Master's thesis, Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1308241476

    Chicago Manual of Style (17th edition)

osu1308241476
427
© 2011, all rights reserved.
This open access ETD is published by The Ohio State University and OhioLINK.