Transfer RNA Recognition Mechanism of Thermoplasma acidophilum Trm56, a SPOUT tRNA Methyltransferase that Possesses an Unusually Long C-terminal Region.

Trm56 from Thermoplasma acidophilum is an exceptional SpoU-TrmD superfamily enzyme, which possesses an unusually long C-terminal region, and catalyzes methylation of 2'-OH of ribose of C56 in tRNA. T. acidophilum Trm56 methylates elongator tRNA (tRNAe) transcript effectively, however this enzyme methylates tRNA transcript slowly. This enzymatic feature is common in T. acidophilum Trm56 and Pyrococcus horikoshii Trm56, which possesses a short C-terminal region. Kinetic analysis revealed that Km value of T. acidophilum Trm56 for tRNA is larger than that for tRNAe. Our analysis of native tRNAs revealed that the 2'-O-methylcytidine content at position 56 (Cm56) in tRNA is 1.9% while in tRNAe it is 63.1%, showing that the in vitro enzymatic properties of T. acidophilum Trm56 are reflected in the Cm56 modification levels in living cells. Experiments with chimera tRNA transcript of tRNA and tRNAe showed that T. acidophilum Trm56 recognizes the T-loop and the three-dimensional core of tRNA. Furthermore, experiments with mutant tRNAe transcript revealed that the G53-C61 base pair and U54U55C56purine57purine58 sequence are essential for methylation. Crystal structures of apo- and 5'-methyl-5'-thioadenosine binding forms of the catalytic domain of T. acidophilum Trm56 revealed that the structure of the 5'-methyl-5'-thioadenosine binding pocket and overall structure of the catalytic domain of T. acidophilum Trm56 closely resemble those of P. horikoshii Trm56. Experiments with a chimera of T. acidophilum and P. horikoshii Trm56 proteins demonstrates that the catalytic domain of T. acidophilum Trm56 is responsible for the slow methylation of tRNA transcript.