Characterization of Molecular Structure-Function Relationships of Escherichia Coli Leucyl-Trna Synthetase
Vu, Michael Tuan
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https://hdl.handle.net/2142/72335
Description
Title
Characterization of Molecular Structure-Function Relationships of Escherichia Coli Leucyl-Trna Synthetase
Author(s)
Vu, Michael Tuan
Issue Date
2008
Doctoral Committee Chair(s)
Martinis, Susan A.
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Abstract
The accurate covalent linkage of amino acid to tRNA for protein synthesis is catalyzed by a family of aminoacyl-tRNA synthetases (aaRS). Some aaRSs have the potential to make mistakes during aminoacylation due to the nature of their amino acid substrate. However, the synthetases have idiosyncratically evolved to include various modules to optimize activity and also enhance fidelity. LeuRS, along with a number of class Ia aaRSs, have acquired a relatively large module called the connective peptide 1 (CP1) domain to correct its mistakes via a hydrolytic amino acid editing mechanism. Surprisingly, deletion of the CP1 domain from Escherichia coli LeuRS yielded a catalytic core that retains fidelity. This suggests that deletion of the CP1 domain may have restored a masked pre-transfer editing mechanism that is inherent in the aminoacylation core.
LeuRS from many organisms has evolved to contain another smaller insert called the leucine-specific (LS) domain. Its function is unclear. Deletion of the LS domain shows that it is important for aminoacylation, but dispensable for amino acid editing. Molecular dissection of the LS domain identified a peptide within the LS domain that impacts enzyme activity. Mutation of a particular aspartate to an alanine residue within the loop dramatically affects the Michaelis constant as well as introduced a biphasic kinetic pattern during aminoacylation under steady state condition. In collaboration, the x-ray crystal structure of the editing complex of E. coli LeuRS was solved. The structure indicates that the aspartate residue is part of a loop within the LS domain, but does not contact the tRNA. It is possible that under the aminoacylation conformation, the LS domain loop could make more intimate contacts with the tRNA.
The x-ray crystal structure also revealed novel tRNA-protein interactions. Biochemical analysis suggests that this interaction is crucial for aminoacylation activity. It is hypothesized that this novel interaction is idiosyncratic to E. coli LeuRS and could serve as an identity element for tRNA selection. We hypothesize that the E. coli LeuRS has evolved to possess unique contacts as well as utilize its inserts to exquisitely interact with the tRNA to enhance fidelity as well as catalytic efficiency.
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