The Elongator subunit Elp3 contains a Fe4S4 cluster and binds S-adenosylmethionine.
The Elp3 subunit of the Elongator complex is highly conserved from archaea to humans and contains a well-characterized C-terminal histone acetyltransferase (HAT) domain. The central region of Elp3 shares significant sequence homology to the Radical SAM superfamily. Members of this large family of bacterial proteins contain a FeS cluster and use S-adenosylmethionine (SAM) to catalyse a variety of radical reactions. To biochemically characterize this domain we have expressed and purified the corresponding fragment of the Methanocaldococcus jannaschii Elp3 protein. The presence of a Fe4S4 cluster has been confirmed by UV-visible spectroscopy and electron paramagnetic resonance (EPR) spectroscopy and the Fe content determined by both a colorimetric assay and atomic absorption spectroscopy. The cysteine residues involved in cluster formation have been identified by site-directed mutagenesis. The protein binds SAM and the binding alters the EPR spectrum of the FeS cluster. Our results provide biochemical support to the hypothesis that Elp3 does indeed contain the Fe4S4 cluster which characterizes the Radical SAM superfamily and binds SAM, suggesting that Elp3, in addition to its HAT activity, has a second as yet uncharacterized catalytic function. We also present preliminary data to show that the protein cleaves SAM.
Paraskevopoulou
Christina
C
Department of Biological Sciences, Imperial College, London, SW7 2BW, UK.
Fairhurst
Shirley A
SA
Lowe
David J
DJ
Brick
Peter
P
Onesti
Silvia
S
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D007501:Iron / Q000737:chemistry; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017015:Methanococcales / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000737:chemistry* / Q000378:metabolism; D013440:Sulfides / Q000737:chemistry
D019843:Archaeal Proteins; D007506:Iron-Sulfur Proteins; D021122:Protein Subunits; D013440:Sulfides; D012436:S-Adenosylmethionine; D007501:Iron; D003545:Cysteine