Identification of Eukaryotic and Prokaryotic Methylthiotransferase for Biosynthesis of 2-Methylthio-N6-threonylcarbamoyladenosine in tRNA

Kazuhito Tomizawa,Mohamed Atta,Simon Arragain,John F. Hunt,Samuel K. Handelman,Marc Fontecave,Fan-Yan Wei,Etienne Mulliez,Farhad Forouhar,Thierry Douki

doi:10.1074/jbc.m110.106831

Abstract

Bacterial and eukaryotic transfer RNAs have been shown to contain hypermodified adenosine, 2-methylthio-N(6)-threonylcarbamoyladenosine, at position 37 (A(37)) adjacent to the 3'-end of the anticodon, which is essential for efficient and highly accurate protein translation by the ribosome. Using a combination of bioinformatic sequence analysis and in vivo assay coupled to HPLC/MS technique, we have identified, from distinct sequence signatures, two methylthiotransferase (MTTase) subfamilies, designated as MtaB in bacterial cells and e-MtaB in eukaryotic and archaeal cells. Both subfamilies are responsible for the transformation of N(6)-threonylcarbamoyladenosine into 2-methylthio-N(6)-threonylcarbamoyladenosine. Recently, a variant within the human CDKAL1 gene belonging to the e-MtaB subfamily was shown to predispose for type 2 diabetes. CDKAL1 is thus the first eukaryotic MTTase identified so far. Using purified preparations of Bacillus subtilis MtaB (YqeV), a CDKAL1 bacterial homolog, we demonstrate that YqeV/CDKAL1 enzymes, as the previously studied MTTases MiaB and RimO, contain two [4Fe-4S] clusters. This work lays the foundation for elucidating the function of CDKAL1.

Highlights

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) BSU25430
Using in vivo gene complementation, we show that proteins from both the MtaB and e-MtaB families are responsible for the conversion of N6-threonylcarbamoyladenosine (t6A) into 2-methylthio-N6-threonylcarbamoyladenosine in tRNA
The third MTTase family identified in our analyses includes yqeV (BSU25430), the other MTTase encoded in the B. subtilis genome, whose enzymatic activity was characterized in this study

Summary

Introduction

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) BSU25430. The MTTase family are two bacterial enzymes as follows: MiaB, which modifies N6-isopentenyladenosine (i6A) to its 2-methylthio derivative (ms2i6A) in tRNA, and RimO, which acts on a specific aspartate residue of the ribosomal S12 protein. Both enzymes have been shown to contain two [4Fe-4S] clusters (4 – 6). The first one is chelated by the three cysteines of a conserved CXXXCXXC motif that is the hallmark signature of the radical AdoMet family (Scheme 1) [7] This cluster serves to bind and reduce AdoMet into methionine and the highly reactive 5Ј-deoxyadenosyl radical (Ado1⁄7) [8]. Bioinformatics analyses presented here demonstrate that five major families of homologous MTTases are encoded in SEPTEMBER 10, 2010 VOLUME 285 NUMBER 37

Methods

Results

Conclusion