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Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors
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Activation of a unique flavin-dependent tRNA-methylating agent
Djemel Hamdane1, Eduardo Bruch, Sun Un
1Laboratoire de Chimie des Processus Biologiques, CNRS-FRE 3488, Collège De France , 11 place Marcelin Berthelot, 75231 Paris Cedex 05, France.
Biochemistry
|November 16, 2013
Summary
TrmFO enzyme uses a unique flavin cofactor to methylate tRNA. Low pH or tRNA addition activates this dormant agent by forming a reactive iminium intermediate, revealing its catalytic mechanism.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- TrmFO is a tRNA methyltransferase utilizing methylenetetrahydrofolate and flavin adenine dinucleotide hydroquinone.
- Previous studies showed TrmFO stabilizes a unique TrmFO-CH2-FADH adduct and a flavin radical.
Purpose of the Study:
- To elucidate the activation mechanism of the dormant methylating agent in TrmFO.
- To characterize the reactive intermediates involved in tRNA methylation.
Main Methods:
- Low pH activation studies.
- Spectroscopic analysis (optical, EPR/ENDOR).
- Density Functional Theory (DFT) calculations.
Main Results:
- TrmFO activation occurs at low pH via protonation of Cys53, cleaving the C-S bond.
- A reactive iminium intermediate, [FADH(N5)═CH2]+, is generated and acts as the methylating agent.
- The flavin radical is a one-electron oxidized product of the adduct, stabilized at neutral/acidic pH and decomposed at high pH.
Conclusions:
- A novel mechanism for flavin-dependent tRNA methylation is proposed, involving a dormant methylating agent activated by pH changes or substrate binding.
- The study provides insights into the structure and reactivity of TrmFO, a unique flavin-based methyltransferase.
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