title: Cysteine of sequence motif VI is essential for nucleophilic catalysis by yeast tRNA m5C methyltransferase.
abstract: Sequence comparison of several RNA m(5)C methyltransferases identifies two conserved cysteine residues that belong to signature motifs IV and VI of RNA and DNA methyltransferases. While the cysteine of motif IV is used as the nucleophilic catalyst by DNA m(5)C methyltransferases, this role is fulfilled by the cysteine of motif VI in Escherichia coli 16S rRNA m(5)C967 methyltransferase, but whether this conclusion applies to other RNA m(5)C methyltransferases remains to be verified. Yeast tRNA m(5)C methyltransferase Trm4p is a multisite-specific S-adenosyl-L-methionine-dependent enzyme that catalyzes the methylation of cytosine at C5 in several positions of tRNA. Here, we confirm that Cys310 of motif VI in Trm4p is essential for nucleophilic catalysis, presumably by forming a covalent link with carbon 6 of cytosine. Indeed, the enzyme is able to form a stable covalent adduct with the 5-fluorocytosine-containing RNA substrate analog, whereas the C310A mutant protein is inactive and unable to form the covalent complex.
authors.affiliation: Laboratoire d'Enzymologie et Biochimie Structurales, Centre National de la Recherche Scientifique, Gif-sur-Yvette Cedex, France.
authors.forename: Hélène
authors.initials: H
authors.lastname: Walbott
authors.forename: Clotilde
authors.initials: C
authors.lastname: Husson
authors.forename: Sylvie
authors.initials: S
authors.lastname: Auxilien
authors.forename: Béatrice
authors.initials: B
authors.lastname: Golinelli-Pimpaneau
mesh_terms: D020816:Amino Acid Motifs; D001483:Base Sequence; D002384:Catalysis; D002467:Cell Nucleus / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry / Q000502:physiology*; D005437:Flucytosine / Q000737:chemistry; D008745:Methylation; D008954:Models, Biological; D050505:Mutant Proteins / Q000378:metabolism; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012359:tRNA Methyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D050505:Mutant Proteins; D029701:Saccharomyces cerevisiae Proteins; D012343:RNA, Transfer; D005437:Flucytosine; D012359:tRNA Methyltransferases; C024845:NCL1 protein, S cerevisiae; D003545:Cysteine