Involved in prephenate-dependent formation of 5-hydroxyuridine (ho5U) modification at position 34 in tRNAs, the first step in 5-carboxymethoxyuridine (cmo5U) biosynthesis (PubMed:31253794). Involved differently in ho5U formation in each tRNA; tRNA(Leu3) and tRNA(Pro3) are major targets of TrhP (PubMed:31253794). Deletion mutant cells display reduced 5-methoxycarbonylmethoxyuridine (mcmo5U) formation in tRNA(Ala1), tRNA(Ser1) and tRNA(Thr4) (PubMed:31253794). Reduced 5-carboxymethoxyuridine (cmo5U) formation in tRNA(Val1) (PubMed:31253794). Strongly reduced cmo5U formation in tRNA(Leu3) and tRNA(Pro3) (PubMed:31253794). Cells lacking both trhP and trhO show complete absence of 5-carboxymethoxyuridine (cmo5U) modification in tRNAs; cells display a temperature-sensitive phenotype and decode codons ending in G (GCG and UCG) less efficiently than the wild-type strain (PubMed:31253794). Belongs to the peptidase U32 family.