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Pseudouridine monophosphate glycosidase: a new glycosidase mechanism.

ABSTRACT: Pseudouridine (?), the most abundant modification in RNA, is synthesized in situ using ? synthase. Recently, a pathway for the degradation of ? 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, ? is first converted to ? 5'-monophosphate (?MP) by ? kinase and then ?MP is degraded by ?MP glycosidase to uracil and ribose 5-phosphate. ?MP 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 ?MP glycosidase and a complex of the K166A mutant with ?MP. We also report the structures of a ring-opened ribose 5-phosphate adduct and a ring-opened ribose ?MP 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 ?MP glycosidase catalyzes the cleavage of the C-C glycosidic bond through a novel ribose ring-opening mechanism.

SUBMITTER: Huang S

PROVIDER: S-EPMC3526674 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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Pseudouridine monophosphate glycosidase: a new glycosidase mechanism.

Huang Siyu S Mahanta Nilkamal N Begley Tadhg P TP Ealick Steven E SE

Biochemistry 20121030 45

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 chara ...[more]

PMID: 23066817

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Project description:BackgroundGlycosidases are involved in several metabolic pathways and the development of inhibitors is an important challenge towards the treatment of diseases, such as diabetes, cancer and viral infections including AIDS. Thus, inhibition of intestinal alpha-glucosidases can be used to treat diabetes through the lowering of blood glucose levels, and alpha-glucosidase inhibitors are being marketed against type 2 (non-insulinodependent mellitus) diabetes (i.e.: Glyset or Diastabol, Basen and Glucor or Precose).ResultsIn that context, new C8-carbasugars and related aminocyclitols have been targeted in order to study the effect of the enhanced flexibility and of the new spatial distribution displayed by these structures on their adaptability in the active site of the enzymes. The synthesis of these new C8-glycomimetics is described from enantiomerically pure C2-symmetrical polyhydroxylated cyclooctenes. Their obtention notably involved a syn-dihydroxylation, and more extended functionalization through formation of a cis-cyclic sulfate followed by amination and subsequent reductive amination. This strategy involving the nucleophilic opening of a cis-cyclic sulfate by sodium azide is to our knowledge the first example in C8-series. It revealead to be an efficient alternative to the nucleoplilic opening of an epoxide moiety which proved unsuccessful in this particular case, due to the hindered conformation of such epoxides as demonstrated by X-ray cristallographic analysis.ConclusionThe biological activity of the synthesized glycomimetics has been evaluated towards 24 commercially available glycosidases. The weak observed activities can probably be related to the spatial disposition of the hydroxy and amino groups which depart too much from that realized in glycomimetics such as valiolamine, voglibose and valienamine. Nevertheless, the synthetic strategy described here is efficient and general, and could be extended to increase the diversity of the glycosidase inhibitors obtained since this diversity is introduced in an ultimate step of the synthesis.

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Pseudouridine monophosphate glycosidase: a new glycosidase mechanism.

Publications

Pseudouridine monophosphate glycosidase: a new glycosidase mechanism.

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