Characterization and Structure of the Aquifex aeolicus Protein DUF752 : A BACTERIAL tRNA-METHYLTRANSFERASE (MnmC2) FUNCTIONING WITHOUT THE USUALLY FUSED OXIDASE DOMAIN (MnmC1) ^*

Abstract

Background: Escherichia coli encodes a bifunctional oxidase/methyltransferase catalyzing the final steps of methylaminomethyluridine (mnm ⁵U) formation in tRNA wobble positions.

Results: Aquifex aeolicus encodes only a monofunctional aminomethyluridine-dependent methyltransferase, lacking the oxidase domain.

Conclusion: An alternative pathway exists for mnm ⁵U biogenesis.

Significance: Information about how an organism modifies the wobble base of its tRNA is important for understanding the emergence of the genetic code.

Abstract

Post-transcriptional modifications of the wobble uridine (U34) of tRNAs play a critical role in reading NNA/G codons belonging to split codon boxes. In a subset of Escherichia coli tRNA, this wobble uridine is modified to 5-methylaminomethyluridine (mnm ⁵U34) through sequential enzymatic reactions. Uridine 34 is first converted to 5-carboxymethylaminomethyluridine (cmnm ⁵U34) by the MnmE-MnmG enzyme complex. The cmnm ⁵U34 is further modified to mnm ⁵U by the bifunctional MnmC protein. In the first reaction, the FAD-dependent oxidase domain (MnmC1) converts cmnm ⁵U into 5-aminomethyluridine (nm ⁵U34), and this reaction is immediately followed by the methylation of the free amino group into mnm ⁵U34 by the S-adenosylmethionine-dependent domain (MnmC2). Aquifex aeolicus lacks a bifunctional MnmC protein fusion and instead encodes the Rossmann-fold protein DUF752, which is homologous to the methyltransferase MnmC2 domain of Escherichia coli MnmC (26% identity). Here, we determined the crystal structure of the A. aeolicus DUF752 protein at 2.5 Å resolution, which revealed that it catalyzes the S-adenosylmethionine-dependent methylation of nm ⁵U in vitro, to form mnm ⁵U34 in tRNA. We also showed that naturally occurring tRNA from A. aeolicus contains the 5-mnm group attached to the C5 atom of U34. Taken together, these results support the recent proposal of an alternative MnmC1-independent shortcut pathway for producing mnm ⁵U34 in tRNAs.