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

Abstract

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 mnm5s2U nucleoside in Escherichia coli tRNAGlu, which are both catalysed by the bifunctional MnmC enzyme. High-performance liquid chromatography-based assays using selectively under-modified tRNA substrates gave a Km value of 600 nM and kcat 0.34 s−1 for the first step, and Km 70 nM and kcat 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 mnm5(s2)U while avoiding build-up of the nm5(s2)U intermediate. The assay method developed here represents a general approach for the comparative analysis of tRNA-modifying enzymes.