Insights into Folate/FAD-dependent tRNA Methyltransferase Mechanism

Abstract

Background:The mechanism of uridine 54 methylation in tRNAs catalyzed by folate/FAD-dependent TrmFO in Bacillus subtilis is unknown. Results: Cys-226 forms a covalent complex with 5-fluorouridine-containing mini-RNA. Conclusion: Thus, Cys-226 acts as the nucleophile instead of Cys-53, located near the active site flavin cofactor. Significance: This third type of folate-dependent uridine methylation mechanism differs from that for thymidylate synthases ThyA and ThyX.

The tRNA Recognition Mechanism of Folate/FAD-dependent tRNA Methyltransferase (TrmFO)

“…the D-arm in the L-shaped tRNA) may hinder the initial binding process. During the course of this study, it has been reported that B. subtilis TrmFO methylates a 31-mer mini-RNA, which includes the T-arm sequence of B. subtilis tRNA Asp (49). The results from our current study are in good agreement with these observations.…”

“…During the course of study, Hamdane et al (49) reported that B. subtilis TrmFO forms a covalent complex with tRNA via Cys-226. According to their report (49), we also attempted to detect the T. thermophilus TrmFO-tRNA complex; however, we unable to observe the formation of the covalent complex by electrophoresis, in which the gel was stained with methylene blue and Coomassie Brilliant Blue (data not shown).…”

“…In contrast, Hamdane et al experimentally verified the covalent bond formation site as Cys-226 in B. subtilis TrmFO (49). Therefore, the covalent bond formation site in our previous hypothetical mechanism should be modified to fit in with the proposal of Hamdane et al (49). If Cys-226 in B. subtilis TrmFO is the covalent bond formation site, TrmFO may form a dimer structure during the enzymatic reaction (49).…”

“…During the course of this study, FAD redox states of B. subtilis TrmFO were reported (48). Furthermore, it has also been reported that B. subtilis TrmFO forms a covalent bond complex with tRNA (48,49). In the current study, we focused on the tRNA recognition mechanisms of T. thermophilus TrmFO.…”

Key steps from the “RNA World” to the “DNA World”

“…covalent linkage between the thiol side chain of an active site cysteine residue and the C6 atom of the targeted base for C5-methylation [16,17]. A similar cysteine-dependent nucleophilic mechanism is also used by S-adenosylmethionine-dependent m5C DNA methyltransferases and RNA methyltransferases that methylate the C5 atom of pyrimidine [18].…”