Glycosidic Bond Conformation Preference Plays a Pivotal Role in Catalysis of RNA Pseudouridylation: A Combined Simulation and Structural Study

摘要

The most abundant chemical modification on RNA is isomerization of uridine, or pseudouridylation, catalyzed by pseudouridine synthases. The catalytic mechanism of this essential process remains largely speculative, partly due to the lack of knowledge of the pre-reactive state that is important to identification of reactive chemical moieties. In the present study, we showed by the orthogonal space random walk free energy simulation method that the pre-reactive states of uridine and its reactive derivative, 5-fluorouridine, bound to a ribonucleoprotein particle (RNP) pseuoduridine synthase strongly prefer the syn glycosidic bond conformation while that of the nonreactive 5-bromouridine-containing substrate is largely populated in the anti conformational state. A high resolution crystal structure of the 5-bromouridine containing substrate bound to the RNP pseuoduridine synthase and enzyme activity assay confirmed the anti nonreactive conformation and provides molecular basis for its confinement. The observed preference for the syn pre-reactive state by the enzyme-bound uridine may help to distinguish among currently proposed mechanisms.