Mechanism of Dihydrouridine Synthase 2 from Yeast and the Importance of Modifications for Efficient tRNA Reduction

Dihydrouridine synthases (DUSs) are flavin-dependent enzymes that catalyze site-specific reduction of uracils in tRNAs. The mechanism of DUS 2 from Saccharomyces cerevisiae was studied. Previously published turnover rates for this DUS were very low. Our studies show that the catalytic cycle consists...

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Bibliographic Details
Published in:The Journal of biological chemistry 2009-04, Vol.284 (16), p.10324-10333
Main Authors: Rider, Lance W., Ottosen, Mette B., Gattis, Samuel G., Palfey, Bruce A.
Format: Article
Language:English
Subjects:
Catalytic Domain
Enzyme Catalysis and Regulation
Escherichia coli
Molecular Structure
NADP - chemistry
NADP - metabolism
Oxidation-Reduction
Oxidoreductases - chemistry
Oxidoreductases - genetics
Oxidoreductases - metabolism
RNA, Transfer, Leu - chemistry
RNA, Transfer, Leu - metabolism
Saccharomyces cerevisiae
Saccharomyces cerevisiae - enzymology
Saccharomyces cerevisiae - genetics
Saccharomyces cerevisiae Proteins - chemistry
Saccharomyces cerevisiae Proteins - genetics
Saccharomyces cerevisiae Proteins - metabolism
Uracil - metabolism
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Summary:Dihydrouridine synthases (DUSs) are flavin-dependent enzymes that catalyze site-specific reduction of uracils in tRNAs. The mechanism of DUS 2 from Saccharomyces cerevisiae was studied. Previously published turnover rates for this DUS were very low. Our studies show that the catalytic cycle consists of reductive and oxidative half-reactions. The enzyme is reduced by NADPH rapidly but has a very slow oxidative half-reaction using in vitro transcribed tRNA substrates. Using tRNALeu purified from a DUS 2 knockout strain of yeast we obtained reaction rate enhancements of 600-fold over in vitro transcribed substrates, indicating that other RNA modifications are required for rapid uracil reduction. This demonstrates a previously unknown ordering of modifications and indicates that dihydrouridine formation is a later step in tRNA maturation. We also show that an active site cysteine is important for catalysis, likely in the protonation of uracil during tRNA reduction. Dihydrouridine of modified tRNA from Escherichia coli was also oxidized to uridine showing the reaction to be reversible.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M806137200