| Abstract
| - Sequence alignment of the TruA, TruB, RsuA, and RluA families of pseudouridine synthases(ΨS) identifies a strictly conserved aspartic acid, which has been shown to be the critical nucleophile forthe ΨS-catalyzed formation of pseudouridine (Ψ). However, superposition of the representative structuresfrom these four families of enzymes identifies two additional amino acids, a lysine or an arginine (K/R)and a tyrosine (Y), from a K/RxY motif that are structurally conserved in the active site. We have createda series of Thermotoga maritima and Escherichia coli pseudouridine 55 synthase (Ψ55S) mutants inwhich the conserved Y is mutated to other amino acids. A new crystal structure of the T. maritima Ψ55SY67F mutant in complex with a 5FU-RNA at 2.4 Å resolution revealed formation of 5-fluoro-6-hydroxypseudouridine (5FhΨ), the same product previously seen in wild-type Ψ55S−5FU-RNA complexstructures. HPLC analysis confirmed efficient formation of 5FhΨ by both Ψ55S Y67F and Y67L mutantsbut to a much lesser extent by the Y67A mutant when 5FU-RNA substrate was used. However, bothHPLC analysis and a tritium release assay indicated that these mutants had no detectable enzymatic activitywhen the natural RNA substrate was used. The combined structural and mutational studies lead us topropose that the side chain of the conserved tyrosine in these four families of ΨS plays a dual role withinthe active site, maintaining the structural integrity of the active site through its hydrophobic phenyl ringand acting as a general base through its OH group for the proton abstraction required in the last step ofΨS-catalyzed formation of Ψ.
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