Mechanistic studies of the pseudouridine synthases TruB and RluA and their interaction with 5-fluorouridine

The mechanism utilized by the pseudouridine synthases (PsiS) to convert uridine to its C-glycoside isomer pseudouridine (Psi) has proven surprisingly elusive. To study this mechanism, RNA with the isomerized uridine replaced by the mechanistic probe 5-fluorouridine (F 5U) have been used. The PsiS TruB handles F5U as a simple substrate and converts F5U into two products when analyzed by reverse-phase HPLC after treatment with S1 nuclease and alkaline phosphatase. After large scale production, mass spectral and NMR analysis of both products revealed they are skeletally identical, isolated as the 5' component of dinucleotides with cytidine and that F5U had become hydrated.;When the PsiS RluA and RNA containing F5U are incubated, they form a stable adduct. After heat disruption and digestion of the freed RNA, reverse-phase HPLC analysis shows only a single F5U product peak, and it elutes distinctly from either product of F5U generated by the action of TruB. The RluA-modified F5U is the 5'-nucleoside of a dinucleotide with the uridine that follows it in the RNA sequence, which neatly explains the difference in retention time compared to the products of TruB. The NMR analysis of the isolated product peak led to the surprising discovery that the single peak contained two co-eluting dinucleotide F 5U products, identical to those formed by TruB except for the expected 3'-nucleoside (U in this case, C with TruB).;The identity of the TruB and RluA F5U products were determined after extensive NMR experiments, complemented by in silico methods. The major F5U product matches that seen in the cocrystal structure of TruB and [F5U]TSL, cis-(5S,6 R)-5,6-dihydro-6-hydroxy-5-fluoropseudouridine. Surprisingly, the minor product is its C2' epimer, cis-(5S,6R)-5,6-dihydro-6-hydroxy-5-fluoropseudouridine-1-beta-D-arabinofuranoside. The discovery that TruB and RluA convert F5U to an arabino minor product raises new and exciting mechanistic possibilities for the PsiS. The most straight forward utilizes the critical aspartate to deprotonate C2', either in a mechanism involving a glycal intermediate, or in a variant of a mechanism with an acylal intermediate that forms a glycal only with the unnatural substrate F5U. This discovery also strongly discredits the mechanism based on Michael addition of the active site aspartate to the nucleobase.