title: Insights into folate/FAD-dependent tRNA methyltransferase mechanism: role of two highly conserved cysteines in catalysis.
abstract: The flavoprotein TrmFO methylates specifically the C5 carbon of the highly conserved uridine 54 in tRNAs. Contrary to most methyltransferases, the 1-carbon unit transferred by TrmFO derives from 5,10-methylenetetrahydrofolate and not from S-adenosyl-L-methionine. The enzyme also employs the FAD hydroquinone as a reducing agent of the C5 methylene U54-tRNA intermediate in vitro. By analogy with the catalytic mechanism of thymidylate synthase ThyA, a conserved cysteine located near the FAD isoalloxazine ring was proposed to act as a nucleophile during catalysis. Here, we mutated this residue (Cys-53 in Bacillus subtilis TrmFO) to alanine and investigated its functional role. Biophysical characterization of this variant demonstrated the major structural role of Cys-53 in maintaining both the integrity and plasticity of the flavin binding site. Unexpectedly, gel mobility shift assays showed that, like the wild-type enzyme, the inactive C53A variant was capable of forming a covalent complex with a 5-fluorouridine-containing mini-RNA. This result confirms the existence of a covalent intermediate during catalysis but rules out a nucleophilic role for Cys-53. To identify the actual nucleophile, two other strictly conserved cysteines (Cys-192 and Cys-226) that are relatively far from the active site were replaced with alanine, and a double mutant C53A/C226A was generated. Interestingly, only mutations that target Cys-226 impeded TrmFO from forming a covalent complex and methylating tRNA. Altogether, we propose a revised mechanism for the m(5)U54 modification catalyzed by TrmFO, where Cys-226 attacks the C6 atom of the uridine, and Cys-53 plays the role of the general base abstracting the C5 proton.
authors.affiliation: Laboratoire d'Enzymologie et Biochimie Structurales, Centre de Recherche de Gif, CNRS, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette, France. hamdane@lebs.cnrs-gif.fr
authors.forename: Djemel
authors.initials: D
authors.lastname: Hamdane
authors.forename: Manuela
authors.initials: M
authors.lastname: Argentini
authors.forename: David
authors.initials: D
authors.lastname: Cornu
authors.forename: Hannu
authors.initials: H
authors.lastname: Myllykallio
authors.forename: Stéphane
authors.initials: S
authors.lastname: Skouloubris
authors.forename: Gaston
authors.initials: G
authors.lastname: Hui-Bon-Hoa
authors.forename: Béatrice
authors.initials: B
authors.lastname: Golinelli-Pimpaneau
mesh_terms: D019943:Amino Acid Substitution; D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000235:genetics / Q000378:metabolism; D008745:Methylation; D020125:Mutation, Missense; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012343:RNA, Transfer / Q000235:genetics / Q000378:metabolism; D012359:tRNA Methyltransferases / Q000235:genetics / Q000276:immunology*
chemical_list: D001426:Bacterial Proteins; D012329:RNA, Bacterial; D005182:Flavin-Adenine Dinucleotide; D012343:RNA, Transfer; D012359:tRNA Methyltransferases