Assay of both activities of the bifunctional tRNA-modifying enzyme MnmC reveals a kinetic basis for selective full modification of cmnm5s2U to mnm5s2U

Nucleic Acids Res. 2011 Jun;39(11):4818-26. doi: 10.1093/nar/gkr071. Epub 2011 Feb 8.


Transfer RNA (tRNA) contains a number of complex 'hypermodified' nucleosides that are essential for a number of genetic processes. Intermediate forms of these nucleosides are rarely found in tRNA despite the fact that modification is not generally a complete process. We propose that the modification machinery is tuned into an efficient 'assembly line' that performs the modification steps at similar, or sequentially increasing, rates to avoid build-up of possibly deleterious intermediates. To investigate this concept, we measured steady-state kinetics for the final two steps of the biosynthesis of the mnm(5)s(2)U nucleoside in Escherichia coli tRNA(Glu), which are both catalysed by the bifunctional MnmC enzyme. High-performance liquid chromatography-based assays using selectively under-modified tRNA substrates gave a K(m) value of 600 nM and k(cat) 0.34 s(-1) for the first step, and K(m) 70 nM and k(cat) 0.31 s(-1) for the second step. These values show that the second reaction occurs faster than the first reaction, or at a similar rate at very high substrate concentrations. This result indicates that the enzyme is kinetically tuned to produce fully modified mnm(5)(s(2))U while avoiding build-up of the nm(5)(s(2))U intermediate. The assay method developed here represents a general approach for the comparative analysis of tRNA-modifying enzymes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromatography, High Pressure Liquid / methods
  • Escherichia coli Proteins / metabolism*
  • Kinetics
  • Multienzyme Complexes / metabolism*
  • RNA, Transfer, Glu / chemistry
  • RNA, Transfer, Glu / metabolism*
  • Thiouridine / analogs & derivatives*
  • Thiouridine / metabolism


  • Escherichia coli Proteins
  • MnmC protein, E coli
  • Multienzyme Complexes
  • RNA, Transfer, Glu
  • Thiouridine
  • 5-methylaminomethyl-2-thiouridine
  • 5-(carboxymethylaminomethyl)-2-thiouridine