Activation of a Unique Flavin-Dependent tRNA-Methylating Agent

详细信息    查看全文

• 作者：Djemel Hamdane ; Eduardo Bruch ; Sun Un ; Martin Field ; Marc Fontecave
• 刊名：Biochemistry
• 出版年：2013
• 出版时间：December 10, 2013
• 年：2013
• 卷：52
• 期：49
• 页码：8949-8956
• 全文大小：383K
• 年卷期：v.52,no.49(December 10, 2013)
• ISSN：1520-4995

文摘

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鈥揅H₂鈥揊ADH adduct and an ill-defined neutral flavin radical. The adduct contains a unique N鈥揅H₂鈥揝 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鈥揝 bond, and generation of a highly reactive [FADH(N⁵)鈺怌H₂]⁺ 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鈥揅H₂鈥揊ADH 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.