Kinetic characterization of substrate-binding sites of thermostable tRNA methyltransferase (TrmB)

TrmB is a eubacterial tRNA methyltransferase which catalyzes the formation of N7-methylguanosine at position 46 (m7G46) in tRNA consuming S-adenosyl-L-methionine (AdoMet) as the methyl group donor during the reaction. Previously, we purified TrmB from Aquifex aeolicus, a hyper-thermophilic eubacterium, and clarified the recognition sites in tRNA. Furthermore, we reported that an additional C-terminal region of A. aeolicus TrmB is required for protein stability at high temperatures. In the current study, we devised a new purification method to remove contaminating RNA completely. The purified enzyme is mainly in a monomeric form. We prepared 17 mutant A. aeolicus TrmB proteins and performed kinetic studies. Our analyses reveal that Glu47, Tyr95, Arg108, Thr165 and Tyr167 residues are important for AdoMet binding and that Asp74, Asp97, and Thr132 are important for the methyltransfer reaction. Furthermore, substitution of Asp133 by alanine caused complete loss of enzymatic activity. Based on the results of our current studies and previous bioinformatic, biochemical and structural studies by others, a reaction mechanism for TrmB is proposed.