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Molecular Basis of Escherichia coli L/F transferase: Catalytic Mechanism and Substrate Specificities

  • Author / Creator
    Fung, Angela Wai Shan
  • The Escherichia coli leucyl/phenylalanyl-tRNA protein transferase (L/F transferase) catalyzes the tRNA-dependent post-translational addition of amino acids onto the N-terminus of a protein polypeptide substrate. The enzymatic N-terminal addition of an amino acid to a protein has been identified as a molecular marker to target proteins for degradation via the N-end rule pathway, where it determines the relationship between the in vivo half-life of a protein and the identity of its N-terminal amino acid. Here we investigate the molecular basis of the catalytic mechanism, substrate analogue design and tRNA substrate recognition by L/F transferase through the analysis of available X-ray crystal structures, mutagenesis, in vitro transcribed tRNAs, and an enzyme functional assay that was developed by our lab (quantitative matrix-assisted laser desorption/ionization time of flight mass spectrometry assay). The N-terminal post-translational addition of an amino acid is analogous to that of peptide bond formation by the ribosome. A previous protein-based catalytic mechanism for L/F transferase has been proposed. Our study on the functional role of D186, a proposed catalytic residue, illustrates that D186’s function is to orient substrates. We propose an alternative substrate-assisted proton shuttling catalytic mechanism, similar to one proposed for the ribosome. The molecular details on tRNA recognition by L/F transferase is studied using crystal structures with tRNA substrate analogues bound, despite differences in their binding orientations. We investigate and illustrate that this difference is due to the different modifications on the analogues. This study leads to the first steps to the design and development of improved substrate analogues for this class of enzyme. Contrary to previous studies suggesting that L/F transferase mainly recognizes the 3' aminoacyl adenosine of an aminoacyl-tRNA for substrate recognition, our studies shed light on the critical importance of recognition of the remaining tRNA body especially the acceptor stem in a sequence-dependent manner. Taken together, our molecular studies into the L/F transferase reaction expand the current understanding of the molecular details of the catalytic mechanism, substrate analogue design, and tRNA substrate recognition for L/F transferase.

  • Subjects / Keywords
  • Graduation date
    2014-11
  • Type of Item
    Thesis
  • Degree
    Doctor of Philosophy
  • DOI
    https://doi.org/10.7939/R3Q81535B
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
    English
  • Institution
    University of Alberta
  • Degree level
    Doctoral
  • Department
    • Department of Biochemistry
  • Supervisor / co-supervisor and their department(s)
    • Fahlman, Richard P.
  • Examining committee members and their departments
    • Kothe, Ute (Chemistry and Biochemistry)
    • Glover, JN Mark (Biochemistry)
    • Owttrim, George (Biological Sciences)
    • Berthiaume, Luc (Cell Biology)