tRNA molecules are extensively tailored containing over 100 of the 110 known RNA base modifications. In addition to expanding the chemical repertoire of tRNAs, posttranscriptional modifications play critical roles in maintaining tRNA structure and function, and their biosynthetic pathways are often very complex. One such biosynthetic pathway is that of the hypermodified base, wybutosine (yW), found adjacent to the wobble position of tRNAPhe in eukaryotes and archaea. The characteristic tricyclic ring of yW is synthesized from methylguanosine (m1G37-tRNAPhe) and a molecule of pyruvate by the S-adenosylmethionine radical enzyme 4-demethylwyosine synthase (TYW1). The mechanism for tricyclic base formation is disputed; two mechanisms have been proposed that differ in activation of pyruvate. In one mechanism, formation of a Schiff base intermediate between pyruvate and a conserved catalytically essential lysine is proposed, activating pyruvate for radical attack by the m1G37-tRNAPhe substrate radical species. In the second mechanism, pyruvate is proposed to be activated by direct ligation to the unique iron of the additional [4Fe-4S] cluster. Here we present biochemical and structural evidence supporting the formation of a Schiff base on pathway to tricyclic ring formation, in agreement with the first mechanism. The crystal structure reveals a Lys-pyruvate Schiff base positioned near the additional [4Fe-4S] cluster. The carboxylate oxygen of pyruvate is ~2.4 Å from the unique iron, favoring a role for the cluster in positioning pyruvate as well as playing a role as an electron sink. Thus, this radical-based addition of two carbon atoms to m1G37-tRNAPhe appears to borrow a page out of a classic enzymology text by employing Schiff base chemistry but with a nearby [4Fe-4S] to facilitate the reaction.

Trapping a Schiff base intermediate in a key tricyclic ring formation step of Wye base biosynthesis

256th National Meeting (2018)

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