Mechanism of dihydrouridine synthase 2 from yeast and the importance of modifications for efficient tRNA reduction

J Biol Chem. 2009 Apr 17;284(16):10324-33. doi: 10.1074/jbc.M806137200. Epub 2009 Jan 12.

Abstract

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 tRNA(Leu) 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Catalytic Domain
  • Molecular Structure
  • NADP / chemistry
  • NADP / metabolism
  • Oxidation-Reduction
  • Oxidoreductases / chemistry
  • Oxidoreductases / genetics
  • Oxidoreductases / metabolism*
  • RNA, Transfer, Leu* / chemistry
  • RNA, Transfer, Leu* / metabolism
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Saccharomyces cerevisiae* / enzymology
  • Saccharomyces cerevisiae* / genetics
  • Uracil / metabolism

Substances

  • RNA, Transfer, Leu
  • Saccharomyces cerevisiae Proteins
  • NADP
  • Uracil
  • Oxidoreductases
  • SMM1 protein, S cerevisiae