A NEW FUNCTION OF S-ADENOSYLMETHIONINE - THE RIBOSYL MOIETY OF ADOMETIS THE PRECURSOR OF THE CYCLOPENTENEDIOL MOIETY OF THE TRANSFER-RNA WOBBLE BASE QUEUINE

Queuosine (Q) 2-cyclopenten-1-yl)amino)methyl)-7-deazaguanosine] usual ly occurs in the first position of the anticodon of tRNAs specifying t he amino acids asparagine, aspartate, histidine, and tyrosine. The hyp ermodified nucleoside is found in eubacteria and eucaryotes. Q is synt hesized de novo exclusively in eubacteria; for eucaryotes the compound is a nutrient factor. In Escherichia coli the Q precursor (oQ), carry ing a 2,3-epoxy-4,5-dihydroxycyclopentane ring, is formed from tRNA pr ecursors containing 7-(aminomethyl)-7-deazaguanine (preQ,) by the queA gene product. A genomic queA mutant accumulating preQ1 tRNA was const ructed. The QueA enzyme was overexpressed as a fusion protein with the glutathione S-transferase from Schistosoma japonicum and purified to homogeneity by affinity and anion-exchange chromatography. The enzyme QueA synthesizes oQ from preQ1 in a single S-adenosylmethionine- (AdoM et-) requiring step, indicating that the ribosyl moiety of AdoMet is t ransferred and isomerized to the epoxycyclopentane residue of oQ. The identity of oQ was verified by HPLC and directly combined HPLC/mass sp ectrometry. The formation of oQ was reconstituted in vitro, applying a synthetic RNA. A 17-nucleotide microhelix (corresponding to the antic odon stem and loop of tRNA(Tyr) from E. coli) is sufficient to act as the RNA substrate for oQ synthesis. We propose that QueA is an S-adeno sylmethionine:tRNA ribosyltransferase-isomerase.