title: A catalytic intermediate and several flavin redox states stabilized by folate-dependent tRNA methyltransferase from Bacillus subtilis.
abstract: The flavoprotein TrmFO catalyzes the C5 methylation of uridine 54 in the TΨC loop of tRNAs using 5,10-methylenetetrahydrofolate (CH(2)THF) as a methylene donor and FAD as a reducing agent. Here, we report biochemical and spectroscopic studies that unravel the remarkable capability of Bacillus subtilis TrmFO to stabilize, in the presence of oxygen, several flavin-reduced forms, including an FADH(•) radical, and a catalytic intermediate endowed with methylating activity. The FADH(•) radical was characterized by high-field electron paramagnetic resonance and electron nuclear double-resonance spectroscopies. Interestingly, the enzyme exhibited tRNA methylation activity in the absence of both an added carbon donor and an external reducing agent, indicating that a reaction intermediate, containing presumably CH(2)THF and FAD hydroquinone, is present in the freshly purified enzyme. Isolation by acid treatment, under anaerobic conditions, of noncovalently bound molecules, followed by mass spectrometry analysis, confirmed the presence in TrmFO of nonmodified FAD. Addition of formaldehyde to the purified enzyme protects the reduced flavins from decay by probably preventing degradation of CH(2)THF. The absence of air-stable reduced FAD species during anaerobic titration of oxidized TrmFO, performed in the absence or presence of added CH(2)THF, argues against their thermodynamic stabilization but rather implicates their kinetic trapping by the enzyme. Altogether, the unexpected isolation of a stable catalytic intermediate suggests that the flavin-binding pocket of TrmFO is a highly insulated environment, diverting the reduced FAD present in this intermediate from uncoupled reactions.
authors.affiliation: Centre de Recherche de Gif, CNRS, 91198 Gif-sur-Yvette, France.
authors.forename: Djemel
authors.initials: D
authors.lastname: Hamdane
authors.forename: Vincent
authors.initials: V
authors.lastname: Guerineau
authors.forename: Sun
authors.initials: S
authors.lastname: Un
authors.forename: Beatrice
authors.initials: B
authors.lastname: Golinelli-Pimpaneau
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology* / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D004578:Electron Spin Resonance Spectroscopy; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005492:Folic Acid / Q000378:metabolism*; D007700:Kinetics; D010084:Oxidation-Reduction; D012343:RNA, Transfer / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013816:Thermodynamics; D012359:tRNA Methyltransferases / Q000737:chemistry* / Q000378:metabolism
chemical_list: D005182:Flavin-Adenine Dinucleotide; D012343:RNA, Transfer; D005492:Folic Acid; D012359:tRNA Methyltransferases