Structural and Biochemical Studies of tRNA Wobble Uridine Hydroxylation

Abstract

tRNAs recognize codons on mRNA and deliver the corresponding amino acids to the ribosome. For accurate decoding, the wobble position of tRNA undergoes post-transcriptional modifications. Among the four nucleotides, uridine exhibits the greatest chemical diversity in its modifications. xo5U-type modifications, which utilize 5-hydroxyuridine as a common intermediate, have been observed in bacteria. However, the structural and biochemical information on the two enzymes responsible for uridine hydroxylation has not been available to date. This dissertation presents the structures of TrhO, an O2-dependent wobble uridine hydroxylase, and investigates the determinants of its activity and the reaction intermediates through biochemical approaches, thereby elucidating its catalytic mechanism.

Chapter I presents the cryo-EM structure of TrhO from Bacillus subtilis in complex with tRNAAla, which reveals that the enzyme primarily interacts with the ASL. Biochemical analyses demonstrate that BsTrhO becomes oxidized and inactivated upon tRNA hydroxylation, and that the catalytic cysteine residue, Cys179, reacts with molecular oxygen without aid of any cofactors. The oxidized BsTrhO can be reduced by reductants such as TCEP or by redox systems such as the Trx system. Based on intact protein MS and DFT calculations, I propose that the catalytic cysteine forms a thio-hydroperoxy intermediate, which transfers the terminal hydroxyl group to U34, forming a sulfenic acid that builds a disulfide bond with Cys185.

Chapter II presents the crystal structure of TrhO from Legionella pneumophila in complex with the ASL region of Bacillus subtilis tRNAAla, which reveals an ASL conformation distinct from that observed in the BsTrhO complex. Additionally, strong electron density for a sulfenic acid was observed at the catalytic cysteine residue, Cys177. Using the same methodology as for BsTrhO, the formation of a disulfide bond was not detected. Finally, I propose that the oxidation of the catalytic cysteine by molecular oxygen and the hydroxyl group transfer process are identical in LpTrhO, and that the final oxidation state of the catalytic cysteine is most likely a sulfenic acid.

Collectively, this dissertation provides structural and biochemical insights into the catalytic mechanism of TrhO from two species. TrhO reacts with molecular oxygen without any cofactors; therefore, its catalytic activity is directly linked to the oxidation state of the catalytic cysteine. These findings provide a new understanding of cofactor-independent monooxygenases and underline the critical role of oxygen in regulating wobble modification.