Steve Ealick's Research Group


Abstract:

Huang S, Mahanta N, Begley TP, and Ealick SE. Pseudouridine Monophosphate Glycosidase: A New Glycosidase Mechanism. Biochemistry 51:9245-9255 (2012).

Pseudouridine (Ψ), the most abundant modification in RNA, is synthesized in situ using Psi synthase. Recently, a pathway for the degradation of Ψ was described (Preumont, A., Snoussi, K., Stroobant, V., Collet, J. F., and Van Schaftingen, E. (2008), J. Biol. Chem. 283, 25238-25246). In this pathway Ψ is first converted to Ψ 5 -monophosphate (ΨMP) by Ψ kinase and then ΨMP is degraded by ΨMP glycosidase to uracil and ribose 5-phosphate. ΨMP glycosidase is the first example of a mechanistically-characterized enzyme that cleaves a C-C glycosidic bond. Here we report X-ray crystal structures of Escherichia coli ΨMP glycosidase and a complex of the K166A mutant with ΨMP. We also report the structures of a ring-opened ribose 5-phosphate adduct and a ring-opened ribose ΨMP adduct. These structures provide four snapshots along the reaction coordinate. The structural studies suggested that the reaction utilizes a Lys166 adduct during catalysis. Biochemical and mass spectrometry data further confirmed the existence of a lysine adduct. We used site directed mutagenesis combined with kinetic analysis to identify roles for specific active site residues. Together, these data suggest that ΨMP glycosidase catalyzes the cleavage of the C-C glycosidic bond through a novel ribose ring-opening mechanism.

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