Stereochemistry of tRNA(m5U54)-methyltransferase catalysis: 19F NMR spectroscopy of an enzyme-FUraRNA covalent complex.

摘要

The catalytic mechanism of tRNA(m5U54)-methyltransferase (RUMT) involves the formation of a covalent adduct between Cys324 of RUMT and C6 of Ura54 in tRNA. The covalent adduct is subsequently methylated at C5 by S-adenosyl-L-methionine (AdoMet). We used an RNA substrate analog containing 5-fluorouracil (FUra) in place of Ura54 to trap the covalent complex and analyzed the adduct by 19F NMR spectroscopy. The 19F NMR spectrum of the adduct consisted of an overlapping doublet of quartets, with an H6-F coupling constant of 4 Hz and a CH3-F coupling constant of 22.4 Hz. On the basis of the magnitude of the H6-F coupling constant, we determined that Cys324 of RUMT and the methyl moiety from AdoMet added across the 5,6-double bond of FUra54 in cis fashion. We deduced that the nucleophilic addition was also cis in the normal enzymatic reaction and that the subsequent beta-elimination of the 5-H and catalytic cysteine was trans. Further, on the basis of chemical considerations, we proposed several conformational adaptations of enzyme-substrate complexes that must occur on the reaction pathway. Together with previous studies, this study enables the proposal of the complete stereochemical pathway for the RUMT-catalyzed methylation of Ura54 in tRNA.