Transfer RNA(5-methylaminomethyl-2-thiouridine)-methyltransferase from Escherichia coli K-12 has two enzymatic activities.

Abstract

The tRNA(5-methylaminomethyl-2-thiouridine)-methyltransferase, which is involved in the biosynthesis of the modified nucleoside 5-methylaminomethyl-2-thiouridine (mnm5s2U) present in the wobble position of some tRNAs, was purified close to homogeneity (95% purity). The molecular mass of the enzyme is 79,000 daltons. The enzyme activity has a pH optimum of 8.0-8.5, is inhibited by magnesium ions, and stimulated by ammonium ions. Two different intermediates in the biosynthesis of mnm5s2U34 are present in tRNA from the mutants trmC1 and trmC2. Unexpectedly, the product present in tRNA from trmC1 cells was identified by mass spectrometric and chromatographic analyses as 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s2U), i.e. a more complex derivative than the final product mnm5s2U. The product present in tRNA from trmC2 cells was identified as 5-aminomethyl-2-thiouridine (nm5s2U). In the presence of S-adenosylmethionine the most purified enzyme fraction converts both cmnm5s2U34 and nm5s2U34 into mnm5s2U34. In the absence of S-adenosylmethionine, however, cmnm5s2U34 is converted into nm5s2U by this enzyme fraction. We conclude that the purified polypeptide has two enzymatic activities; one actually demodifies cmnm5s2U to nm5s2U and the other catalyzes the transfer of a methyl group from S-adenosylmethionine to nm5s2U, thus forming mnm5s2U. The sequential order of the biosynthesis of mnm5s2U34 is suggested to be: (Formula: see text). The molecular activity of the methyltransferase activity (nm5s2U34----mnm5s2U34) is 74 min-1, and the steady state concentration of the enzyme is only 78 molecules/genome equivalent in cells growing at a specific growth rate of 1.0/h.