5 years ago

Structural and biochemical insights into the 2′-O-methylation of pyrimidines 34 in tRNA

Structural and biochemical insights into the 2′-O-methylation of pyrimidines 34 in tRNA
Xiangyu Deng, Zhong Wang, Panjiao Pang, Wei Xie
tRNA molecules undergo extensive modifications during their maturation and these modifications play important cellular roles. TrmL is a tRNA-modification enzyme that catalyzes the transfer of a methyl group to the 2′-hydroxyl group of the pyrimidines at the wobble position 34 in two tRNALeu isoacceptors, but the mechanism remains elusive. In this study, we determined the crystal structure of TrmL from Thermus thermophilus (TtTrmL) to 1.7 Å. The enzyme contains only the conserved minimal SPOUT fold, but displays distinct biochemical behavior from its Escherichia coli counterpart, EcTrmL. Interestingly, a fortuitous ligand of 5′-methylthioadenosine was consistently found at the SAM-binding pocket in the crystal structures, which probably came from the expression host. Both TtTrmL and EcTrmL were capable of methylating each other's tRNA substrates, but the latter exhibited much higher activity than the former. Enzymatic activity assays showed that the reaction catalyzed by TtTrmL greatly depends on the reaction pH and is also affected by salt concentration. Via sequence alignment and structural superposition, we discovered that a universally conserved glutamate residue is likely to fulfill the role of the general base for the initial proton abstraction from the 2′-hydroxyl group of pyrimidines 34. Lastly, based on our structural and biochemical data, we proposed the dimer interface to be the tRNA-binding site for TtTrmL. Database The atomic coordinates and structural factors have been deposited in the Protein Data Bank with accession number 5CO4. TrmL is a tRNA methyltransferase that methylates the 2′-hydroxyl group of two leucine tRNAs at the wobble position 34. We determined the crystal structure of TrmL from Thermus thermophilus bound by a fortuitous ligand of 5′-methylthioadenosine (MTA), which allowed us to reveal the catalytic mechanism and the tRNA-binding site of the enzyme (dimer interface).

Publisher URL: http://onlinelibrary.wiley.com/resolve/doi

DOI: 10.1111/febs.14120

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