Pseudouridine monophosphate glycosidase: a new glycosidase mechanism.

Pseudouridine (Ψ) was the first modified nucleoside to be discovered and is the most abundant modification in RNA, existing in tRNAs, rRNAs, snRNAs and snoRNAs (1). Ψ is biosynthesized post-transcriptionally from RNA uridine moieties by Ψ synthase, which cleaves the glycosidic C-N bond and reconnects the uracil to the ribosyl moiety at the C5 position (2). Pseudouridine reduces the conformational flexibility of RNA because the exposed, protonated N1 atom forms strong hydrogen bonds with structured water molecules, replacing weak interactions formed by C5 (1, 3). While Ψ biosynthesis has been studied extensively, little is known about Ψ catabolism. Recently, the two enzymes responsible for Ψ 1 degradation in Escherichia coli were identified (4). Ψ kinase first phosphorylates Ψ to pseudouridine 5′-phosphate (ΨMP, 2). ΨMP glycosidase then catalyzes the reversible cleavage of the C-C glycosidic bond to form uracil 4 and ribose 5-phosphate (R5P, 3) (Scheme 1). While ΨMP glycosidase functions biologically in the cleavage direction, equilibrium strongly favors ΨMP synthesis with an equilibrium constant for the degradation reaction of 2.3 × 10-4 M (4). In bacterial genomes Ψ kinase and ΨMP glycosidase are encoded by separate genes. In E. coli these genes are yeiC and yeiN, respectively, which are located in the same operon. In some eukaryotes, the genes for Ψ kinase and ΨMP glycosidase are fused, resulting in a bifunctional enzyme. Enzymes metabolizing Ψ are not found in humans and most other higher organisms, and excess Ψ is excreted (5). Scheme 1 While natural products containing O and N glycosidic bonds are widespread, natural products containing the C-glycosidic bond are much less prevalent (6). The C-glycosidic bond is generally assumed to be formed by an electrophilic aromatic substitution. Enzymes that cleave C-N glycosidic bonds have been extensively studied. These include nucleoside and nucleotide hydrolases (7-9), purine and pyrimidine nucleoside phosphorylases (10), purine and pyrimidine phosphoribosyltransferases (11, 12), and nucleoside deoxyribosyltransferases (13). In addition, DNA-repair enzymes such as endonuclease III function as N-glycosylases to excise damaged nucleobases from DNA (14). Enzymes that cleave C-O glycosidic bonds are widespread in carbohydrate metabolism and have also been extensively studied (15). Biochemical, biophysical and genetic studies suggest that these enzymes cleave C-N and C-O glycosidic bonds by a largely dissociative mechanism generating an oxocarbenium ion intermediate. While the genes encoding several C-glycosynthases have been identified and in many cases overexpressed (16-23) – and the structures of ligand free UrdGT2 (24, 25) and ΨMP glycosidase (26) have been determined – there is currently no example of a mechanistically well-characterized C-glycosidase or glycosynthase. Here we report a set of E. coli ΨMP glycosidase (EcΨMP glycosidase) crystal structures, which provide four snapshots of the reaction coordinate. The structures are: the unliganded enzyme, a ΨMP glycosidase/ring-opened R5P adduct, a ΨMP glycosidase/ ring-opened ribose ΨMP adduct, and a K166A ΨMP complex. The structural studies suggested that the reaction involves an intermediate imine with Lys166. This was confirmed by mass spectrometry. Kinetic studies on active site mutants suggested roles for individual amino acid side chains. Comparison of the EcΨMP glycosidase structures suggested a role for conformational changes in ΨMP cleavage and product release. These results suggest an unanticipated mechanism for the pseudouridine glycosidase catalyzed C-glycosyl bond hydrolysis.