Mechanistic investigations of the pseudouridine synthase RluA using RNA containing 5-fluorouridine.

The pseuoduridine synthases (Ψ synthases) isomerize uridine (U) to pseudouridine (Ψ) in RNA, and they fall into five families that share very limited sequence similarity but have the same overall fold and active-site architecture, including an essential Asp. The mechanism by which the Ψ synthases operate remains unknown, and mechanistic work has largely made use of RNA containing 5-fluorouridine (f5U) in place of U. The Ψ synthase TruA forms a covalent adduct with such RNA, and heat disruption of the adduct generates a hydrated product of f5U, which was reasonably concluded to result from the hydrolysis of an ester linkage between the essential Asp and f5U. In contrast, the Ψ synthase TruB, which is a member of a different family, does not form an adduct with f5U in RNA but catalyzes the rearrangement and hydration of the f5U, which labeling studies with [18O]water showed does not result from ester hydrolysis. To extend the line of mechanistic investigation to another family of Ψ synthases and an enzyme t...