tRNA modification by S-adenosylmethionine:tRNA ribosyltransferase-isomerase -: Assay development and characterization of the recombinant enzyme

The enzyme S-adenosylmethionine:tRNA ribosyltransferase-isomerase catalyzes the penultimate step in the biosynthesis of the hypermodified tRNA nucleoside queuosine (Q), an unprecedented ribosyl transfer from the cofactor S-adenosylmethionine (AdoMet) to a modified-tRNA precursor to generate epoxyqueuosine (oQ). The complexity of the reaction makes it an especially interesting mechanistic problem, and as a foundation for detailed kinetic and mechanistic studies we have carried out the basic characterization of the enzyme. Importantly, to allow for the direct measurement of oQ formation, we have developed protocols for the preparation of homogeneous substrates; specifically, an overexpression system was constructed for tRNATyr in an E. coli queA deletion mutant to allow for the isolation of large quantities of substrate tRNA, and [U-ribosyl-C-14]AdoMet was synthesized. The enzyme shows optimal activity at pH 8.7 in buffers containing various oxyanions, including acetate, carbonate, EDTA, and phosphate. Unexpectedly, the enzyme was inhibited by Mg2+ and Mn2+ in millimolar concentrations. The steady-state kinetic parameters were determined to be K-m(AdoMet) = 101.4 muM, K-m(tRNA) = 1.5 muM, and k(cat) = 2.5 min(-1). A short minihelix RNA was synthesized and modified with the precursor 7-aminomethyl-7-deazaguanine, and this served as an efficient substrate for the enzyme (K-m(RNA) = 37.7 muM and k(cat) = 14.7 min(-1)), demonstrating that the anticodon stem-loop is sufficient for recognition and catalysis by QueA.