title: 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.
abstract: 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.
authors.affiliation: Laboratoire de Chimie et Biologie des Métaux, équipe Biocatalyse, Institut de Recherches en Technologies et Sciences pour le Vivant, iRTSV-LCBM/Biocat, UMR 5249 Commissariat à l'Energie Atomique (CEA)/CNRS/Université Joseph Fourier (UJF), CEA/Grenoble, 17, rue des Martyrs, 38054 Grenoble Cedex 09, France.
authors.forename: Phanélie
authors.initials: P
authors.lastname: Perche-Letuvée
authors.forename: Velavan
authors.initials: V
authors.lastname: Kathirvelu
authors.forename: Gustav
authors.initials: G
authors.lastname: Berggren
authors.forename: Martin
authors.initials: M
authors.lastname: Clemancey
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Latour
authors.forename: Vincent
authors.initials: V
authors.lastname: Maurel
authors.forename: Thierry
authors.initials: T
authors.lastname: Douki
authors.forename: Jean
authors.initials: J
authors.lastname: Armengaud
authors.forename: Etienne
authors.initials: E
authors.lastname: Mulliez
authors.forename: Marc
authors.initials: M
authors.lastname: Fontecave
authors.forename: Ricardo
authors.initials: R
authors.lastname: Garcia-Serres
authors.forename: Serge
authors.initials: S
authors.lastname: Gambarelli
authors.forename: Mohamed
authors.initials: M
authors.lastname: Atta
mesh_terms: D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D002262:Carboxy-Lyases / Q000235:genetics / Q000502:physiology*; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D016000:Cluster Analysis; D003545:Cysteine / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D006151:Guanosine / Q000031:analogs & derivatives / Q000737:chemistry; D007506:Iron-Sulfur Proteins / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics / Q000502:physiology*; D043903:Pyrococcus abyssi / Q000201:enzymology* / Q000235:genetics; D019289:Pyruvic Acid / Q000737:chemistry; D012343:RNA, Transfer / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014466:Ultraviolet Rays
chemical_list: D019843:Archaeal Proteins; D007506:Iron-Sulfur Proteins; D029701:Saccharomyces cerevisiae Proteins; D006151:Guanosine; C012100:wyosine; D012436:S-Adenosylmethionine; D019289:Pyruvic Acid; D012343:RNA, Transfer; D010088:Oxidoreductases; C569111:TYW1 protein, S cerevisiae; C578091:4-demethylwyosine synthase, Pyrococcus abyssi; D002262:Carboxy-Lyases; D003545:Cysteine