Enzymatic characterization and mutational studies of TruD – the fifth family of pseudouridine synthases

Pseudouridine (Ψ) is formed through isomerization of uridine (U) catalyzed by a class of enzymes called pseudouridine synthases (ΨS). TruD is the fifth family of ΨS. Studies of the first four families (TruA, TruB, RsuA, and RluA) of ΨS reveal a conserved Asp and Tyr are critical for catalysis. However, in TruD family, the tyrosine is not conserved. In this study, we measured the enzymatic parameters for TruD in Escherichia coli, and carried out enzymatic assays for a series of single, double, and triple TruD mutants. Our studies indicate that a Glu, strictly conserved in only TruD family is likely to be the general base in TruD. We also proposed a possible distinct mechanism of TruD-catalyzed Ψ formation compared to the first four families.