Insights into folate/FAD-dependent tRNA methyltransferase mechanism: role of two highly conserved cysteines in catalysis

J Biol Chem. 2011 Oct 21;286(42):36268-80. doi: 10.1074/jbc.M111.256966. Epub 2011 Aug 16.

Abstract

The flavoprotein TrmFO methylates specifically the C5 carbon of the highly conserved uridine 54 in tRNAs. Contrary to most methyltransferases, the 1-carbon unit transferred by TrmFO derives from 5,10-methylenetetrahydrofolate and not from S-adenosyl-L-methionine. The enzyme also employs the FAD hydroquinone as a reducing agent of the C5 methylene U54-tRNA intermediate in vitro. By analogy with the catalytic mechanism of thymidylate synthase ThyA, a conserved cysteine located near the FAD isoalloxazine ring was proposed to act as a nucleophile during catalysis. Here, we mutated this residue (Cys-53 in Bacillus subtilis TrmFO) to alanine and investigated its functional role. Biophysical characterization of this variant demonstrated the major structural role of Cys-53 in maintaining both the integrity and plasticity of the flavin binding site. Unexpectedly, gel mobility shift assays showed that, like the wild-type enzyme, the inactive C53A variant was capable of forming a covalent complex with a 5-fluorouridine-containing mini-RNA. This result confirms the existence of a covalent intermediate during catalysis but rules out a nucleophilic role for Cys-53. To identify the actual nucleophile, two other strictly conserved cysteines (Cys-192 and Cys-226) that are relatively far from the active site were replaced with alanine, and a double mutant C53A/C226A was generated. Interestingly, only mutations that target Cys-226 impeded TrmFO from forming a covalent complex and methylating tRNA. Altogether, we propose a revised mechanism for the m(5)U54 modification catalyzed by TrmFO, where Cys-226 attacks the C6 atom of the uridine, and Cys-53 plays the role of the general base abstracting the C5 proton.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Bacillus subtilis / enzymology*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Flavin-Adenine Dinucleotide / genetics
  • Flavin-Adenine Dinucleotide / metabolism
  • Methylation
  • Mutation, Missense
  • RNA, Bacterial / genetics
  • RNA, Bacterial / metabolism
  • RNA, Transfer / genetics
  • RNA, Transfer / metabolism
  • tRNA Methyltransferases / genetics
  • tRNA Methyltransferases / immunology*

Substances

  • Bacterial Proteins
  • RNA, Bacterial
  • Flavin-Adenine Dinucleotide
  • RNA, Transfer
  • tRNA Methyltransferases