Pseudouridine monophosphate glycosidase: a new glycosidase mechanism.
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) 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.
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, United States.
Siyu
S
Huang
Nilkamal
N
Mahanta
Tadhg P
TP
Begley
Steven E
SE
Ealick
D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D006026:Glycoside Hydrolases; C578388:pseudouridine monophosphate glycosidase, E coli; D008239:Lysine