Pseudouridine Monophosphate
Glycosidase: A New Glycosidase
Mechanism
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Abstract
Pseudouridine (Ψ), the most abundant modification
in RNA,
is synthesized in situ using Ψ 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) <i>J. Biol. Chem. 283</i>, 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 <i>Escherichia coli</i> 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