期刊
JOURNAL OF MOLECULAR BIOLOGY
卷 401, 期 5, 页码 690-695出版社
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2010.06.061
关键词
pseudouridine synthase; H/ACA ribonucleoprotein; free energy simulation; catalytic mechanism; ring conformation
资金
- National Institutes of Health [R01 GM66958-01]
- National Science Foundation [0919983]
- American Heart Association Florida/Puerto Rico [0815267E]
- US Department of Energy, Office of Science, Office of Basic Energy Sciences [W-31-109-Eng-38]
- Direct For Biological Sciences
- Div Of Molecular and Cellular Bioscience [0919983] Funding Source: National Science Foundation
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 lack of knowledge of the pre-reactive state that is important to the identification of reactive chemical moieties. In the present study, we showed, using orthogonal space random-walk free-energy simulation, that the pre-reactive states of uridine and its reactive derivative 5-fluorouridine, bound to a ribonucleoprotein particle pseudouridine synthase, strongly prefer the syn glycosidic bond conformation, while that of the nonreactive 5-bromouridine-containing substrate is largely populated in the anti conformation state. A high-resolution crystal structure of the 5-bromouridine-containing substrate bound to the ribonucleoprotein particle pseudouridine synthase and enzyme activity assay confirmed the anti nonreactive conformation and provided the 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. Published by Elsevier Ltd.
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