Threonyl tRNA synthetases as antibiotic targets and resistance mechanisms

Liston, Jonathon (2022) Threonyl tRNA synthetases as antibiotic targets and resistance mechanisms. Doctoral thesis, University of East Anglia.

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Abstract

Threonyl tRNA synthetases (ThrRSs) catalyse the attachment of L-threonine to its cognate tRNAThr. These enzymes are essential for the translation of proteins. There are currently two known natural product ThrRS inhibitors, borrelidin and obafluorin, produced by Streptomyces parvulus Tü 4055 and Pseudomonas fluorescens ATCC 39502 respectively. Both of their biosynthetic gene clusters (BGCs) encode secondary ThrRSs which provide self-resistance mechanisms. In the borrelidin BGC, this is BorO and in the obafluorin BGC, this is ObaO. While the biosynthesis of both compounds is well understood, the mechanisms of self-resistance are not, and the mechanism of action of obafluorin remains elusive.

Here the structure of BorO was solved and it was found that both ThrRSs in the producer, S. parvulus (Sp), are resistant to borrelidin. Mutagenesis of the Escherichia coli target, EcThrRS, identified a L489T mutation which is sufficient to confer resistance by preventing borrelidin binding, explaining resistance by BorO. In SpThrRS, this residue is a glutamate and an EcThrRS, L489Q mutation is not sufficient to confer resistance, meaning that SpThrRS has a distinct borrelidin resistance mechanism. It was unexpectedly found that ObaO can confer resistance to borrelidin due to the presence of a methionine in this same position and the EcThrRS L489M mutant is resistant to borrelidin. Introduction of this mutation allowed the first structure of ObaO to be solved by X-ray crystallography. Cryogenic electron microscopy (Cryo-EM) structures of obafluorin bound to both EcThrRS and ObaO were solved, showing that EcThrRS covalently links to obafluorin through Y462, while the interaction is non-covalent for ObaO. Spontaneous resistant mutagenesis identified the serine (S463) immediately adjacent to Y462as an essential component of the ObaO resistance to obafluorin.

Finally, a survey of published genomes for additional copies of ThrRS identified BGCs encoding potentially novel natural product ThrRS inhibitors.

Item Type:	Thesis (Doctoral)
Faculty \ School:	Faculty of Science > School of Chemistry
Depositing User:	Chris White
Date Deposited:	24 Apr 2023 14:08
Last Modified:	24 Apr 2023 14:08
URI:	https://ueaeprints.uea.ac.uk/id/eprint/91869
DOI:

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