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

Descriptions

Other title
Subject/Keyword
L/F transferase
post-translational addition of amino acids
N-end rule
non-ribosomal peptide bond formation
Dupli-GNAT superfamily
aminoacyl-tRNA
N-degrons
regulated proteolysis
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Fung, Angela Wai Shan
Supervisor and department
Fahlman, Richard P.
Examining committee member and department
Glover, JN Mark (Biochemistry)
Berthiaume, Luc (Cell Biology)
Owttrim, George (Biological Sciences)
Kothe, Ute (Chemistry and Biochemistry)
Department
Department of Biochemistry
Specialization

Date accepted
2014-09-12T11:18:56Z
Graduation date
2014-11
Degree
Doctor of Philosophy
Degree level
Doctoral
Abstract
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.
Language
English
DOI
doi:10.7939/R3Q81535B
Rights
This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for the purpose of private, scholarly or scientific research. 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.
Citation for previous publication
AW Fung, HA Ebhardt, H Abeysundara, J Moore, Z Xu, and RP Fahlman (2011) An Alternative Mechanism for the Catalysis of Peptide Bond Formation by L/F Transferase: Substrate Binding and Orientation. Journal of Molecular Biology. 409 (4): 617-629.AW Fung, HA Ebhardt, KS Krishnakumar, J Moore, Z Xu, P Strazewski, and RP Fahlman (2014) Probing the Leucyl/Phenylalanyl tRNA Protein Transferase Active Site with tRNA Substrate Analogues. Protein and Peptide Letters. 21 (7): 603-614.AW Fung, CC Leung and RP Fahlman (2014) The Determination of tRNALeu Recognition Nucleotides for Escherichia coli L/F transferase. RNA. 20 (8): 1210-1222.

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File title: Table of Contents.pdf
File title: Angela Wai Shan Fung
File author: Angela Fung
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