4-Demethylwyosine synthase from Pyrococcus abyssi is a radical-S-adenosyl-L-methionine enzyme with an additional [4Fe-4S](+2) cluster that interacts with the pyruvate co-substrate

J Biol Chem. 2012 Nov 30;287(49):41174-85. doi: 10.1074/jbc.M112.405019. Epub 2012 Oct 5.


Wybutosine and its derivatives are found in position 37 of tRNA encoding Phe in eukaryotes and archaea. They are believed to play a key role in the decoding function of the ribosome. The second step in the biosynthesis of wybutosine is catalyzed by TYW1 protein, which is a member of the well established class of metalloenzymes called "Radical-SAM." These enzymes use a [4Fe-4S] cluster, chelated by three cysteines in a CX(3)CX(2)C motif, and S-adenosyl-L-methionine (SAM) to generate a 5'-deoxyadenosyl radical that initiates various chemically challenging reactions. Sequence analysis of TYW1 proteins revealed, in the N-terminal half of the enzyme beside the Radical-SAM cysteine triad, an additional highly conserved cysteine motif. In this study we show by combining analytical and spectroscopic methods including UV-visible absorption, Mössbauer, EPR, and HYSCORE spectroscopies that these additional cysteines are involved in the coordination of a second [4Fe-4S] cluster displaying a free coordination site that interacts with pyruvate, the second substrate of the reaction. The presence of two distinct iron-sulfur clusters on TYW1 is reminiscent of MiaB, another tRNA-modifying metalloenzyme whose active form was shown to bind two iron-sulfur clusters. A possible role for the second [4Fe-4S] cluster in the enzyme activity is discussed.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Archaeal Proteins / chemistry*
  • Archaeal Proteins / genetics*
  • Archaeal Proteins / physiology
  • Carboxy-Lyases / genetics
  • Carboxy-Lyases / physiology*
  • Catalysis
  • Chromatography, High Pressure Liquid
  • Cloning, Molecular
  • Cluster Analysis
  • Cysteine / genetics
  • Electron Spin Resonance Spectroscopy
  • Guanosine / analogs & derivatives
  • Guanosine / chemistry
  • Iron-Sulfur Proteins / chemistry
  • Mass Spectrometry / methods
  • Models, Chemical
  • Molecular Sequence Data
  • Oxidoreductases / genetics
  • Oxidoreductases / physiology*
  • Pyrococcus abyssi / enzymology*
  • Pyrococcus abyssi / genetics
  • Pyruvic Acid / chemistry
  • RNA, Transfer / metabolism
  • S-Adenosylmethionine / genetics
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / physiology*
  • Sequence Homology, Amino Acid
  • Substrate Specificity
  • Ultraviolet Rays


  • Archaeal Proteins
  • Iron-Sulfur Proteins
  • Saccharomyces cerevisiae Proteins
  • Guanosine
  • wyosine
  • S-Adenosylmethionine
  • Pyruvic Acid
  • RNA, Transfer
  • Oxidoreductases
  • TYW1 protein, S cerevisiae
  • 4-demethylwyosine synthase, Pyrococcus abyssi
  • Carboxy-Lyases
  • Cysteine