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

Pearson, David, Carell, Thomas
Nucleic acids research 2011 v.39 no.11 pp. 4818-4826
Escherichia coli, biosynthesis, enzymes, nucleosides, transfer RNA
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.