Activation of a Unique Flavin-Dependent tRNA-Methylating Agent

Abstract : TrmFO is a tRNA methyltransferase that uses methylenetetrahydrofolate (CH2THF) and flavin adenine dinucleotide hydroquinone as cofactors. We have recently shown that TrmFO from Bacillus subtilis stabilizes a TrmFO-CH2-FADH adduct and an ill-defined neutral flavin radical. The adduct contains a unique N-CH2-S moiety, with a methylene group bridging N5 of the isoalloxazine ring and the sulfur of an active-site cysteine (Cys53). In the absence of tRNA substrate, this species is remarkably stable but becomes catalytically competent for tRNA methylation following tRNA addition using the methylene group as the source of methyl. Here, we demonstrate that this dormant methylating agent can be activated at low pH, and we propose that this process is triggered upon tRNA addition. The reaction proceeds via protonation of Cys53, cleavage of the C-S bond, and generation of a highly reactive [FADH(N5)═CH2]+ iminium intermediate, which is proposed to be the actual tRNA-methylating agent. This mechanism is fully supported by DFT calculations. The radical present in TrmFO is characterized here by optical and EPR/ENDOR spectroscopy approaches together with DFT calculations and is shown to be the one-electron oxidized product of the TrmFO-CH2-FADH adduct. It is also relatively stable, and its decomposition is facilitated by high pH. These results provide new insights into the structure and reactivity of the unique flavin-dependent methylating agent used by this class of enzymes.
Type de document :
Article dans une revue
Biochemistry, American Chemical Society, 2013, 52 (49), pp.8949 - 8956. 〈10.1021/bi4013879〉
Liste complète des métadonnées

http://hal.univ-grenoble-alpes.fr/hal-01662525
Contributeur : Frank Thomas <>
Soumis le : mercredi 13 décembre 2017 - 11:23:22
Dernière modification le : jeudi 28 juin 2018 - 09:51:21

Identifiants

Collections

Citation

Marc Hamdane, Eduardo Bruch, Sun Un, Martin J Field, Marc Fontecave. Activation of a Unique Flavin-Dependent tRNA-Methylating Agent. Biochemistry, American Chemical Society, 2013, 52 (49), pp.8949 - 8956. 〈10.1021/bi4013879〉. 〈hal-01662525〉

Partager

Métriques

Consultations de la notice

103