Functional characterization of the YmcB and YqeV tRNA methylthiotransferases of Bacillus subtilis

Abstract

Methylthiotransferases (MTTases) are a closely related family of proteins that perform both radical-S-adenosylmethionine (SAM) mediated sulfur insertion and SAM-dependent methylation to modify nucleic acid or protein targets with a methyl thioether group (–SCH3). Members of two of the four known subgroups of MTTases have been characterized, typified by MiaB, which modifies N6-isopentenyladenosine (i6A) to 2-methylthio-N6-isopentenyladenosine (ms2i6A) in tRNA, and RimO, which modifies a specific aspartate residue in ribosomal protein S12. In this work, we have characterized the two MTTases encoded by Bacillus subtilis 168 and find that, consistent with bioinformatic predictions, ymcB is required for ms2i6A formation (MiaB activity), and yqeV is required for modification of N6-threonylcarbamoyladenosine (t6A) to 2-methylthio-N6-threonylcarbamoyladenosine (ms2t6A) in tRNA. The enzyme responsible for the latter activity belongs to a third MTTase subgroup, no member of which has previously been characterized. We performed domain-swapping experiments between YmcB and YqeV to narrow down the protein domain(s) responsible for distinguishing i6A from t6A and found that the C-terminal TRAM domain, putatively involved with RNA binding, is likely not involved with this discrimination. Finally, we performed a computational analysis to identify candidate residues outside the TRAM domain that may be involved with substrate recognition. These residues represent interesting targets for further analysis.

Diversity of the biosynthesis pathway for threonylcarbamoyladenosine (t6A), a universal modification of tRNA

“…YqeV (MtaB) in B. subtilis and Cdkal1 in humans are responsible for the insertion of the sulfur moiety and methylation at position 2 of the adenosine containing t 6 A. [65][66] MtaB has been shown to increase the accuracy of decoding lysine codons. 66,67 Loss of the Cdkal1 homolog in mice is correlated to increase Type 2 diabetes.…”

Glycyl radical activating enzymes: Structure, mechanism, and substrate interactions

“…These auxiliary Fe–S clusters have become a rising theme in radical SAM enzymes [2]. These additional cluster(s) have been shown to facilitate sulfur insertion into product [48–50] or to assist in methylthiotransferase reactions as the sulfur source [51–53]. Other possible purposes for auxiliary Fe–S clusters include reducing the radical SAM [4Fe–4S] cluster [54], acting as an electron acceptor during catalysis [55], or substrate coordination [56].…”

An updated metabolic view of the Bacillus subtilis 168 genome

“…Besides the previously identified features of macromolecule modifications, the biosynthesis of themodifiednucleotide2-methylthio-N6-threocarbamoyladenosine, a modified residue at position 37, adjacent to the 39-end of the anticodon of several tRNAs has been characterized (MicrorefCyc, PWYIPS-51). This complex modification, which is found in Firmicutes and extends that found in gamma-proteobacteria, combines synthesis and transfer of the threonylcarbamoyl-group to the N6 atom of the adenine residue at position 37 of tRNA catalysed by a complex of four proteins, TsaC (YwlC, RimN), TsaE (YdiB), TsaB (YdiC) and TsaD (YdiE) tRNA threonylcarbamoyltransferase, and then the transfer of methylthiol group to threonylcarbamoyladenosine 37 by the class 3 methylthiotransferase MtaB (YqeV, RimO) (Anton et al, 2008(Anton et al, , 2010Arragain et al, 2010;Lauhon, 2012).…”