tRNA:m(5)C methyltransferase Trm4 generates the modified nucleotide 5-methylcytidine in archaeal and eukaryotic tRNA molecules, using S-adenosyl-L-methionine (AdoMet) as the methyl donor. Most Archaea and Eukaryotes possess several Trm4 homologs, including those related to diseases, while the archaeon Methanocaldococcus jannaschii has only one gene encoding a Trm4 homolog, MJ0026. The recombinant MJ0026 protein catalyzed the AdoMet-dependent methyltransferase activity on tRNA in vitro, and was shown to be the M. jannaschii Trm4. We determined the crystal structures of the substrate-free M. jannaschii Trm4 and its complex with sinefungin at 1.27 A and 2.3 A resolutions, respectively. This AdoMet analog is bound in a negatively-charged pocket near helix alpha8. This helix can adopt two different conformations, thereby controlling the entry of the AdoMet into the active site. Adjacent to the sinefungin-bound pocket, highly conserved residues form a large, positively-charged surface, which seems to be suitable for tRNA binding. The structure explains the roles of several conserved residues that were reportedly involved in the enzymatic activity or stability of Trm4p from the yeast Saccharomyces cerevisiae. We also discuss the previous genetic and biochemical data of the human NSUN2/hTrm4/Misu and the archaeal PAB1947 methyltransferase, based on the structure of M. jannaschii Trm4.CI - Copyright (c) 2010. Published by Elsevier Ltd.
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