Functional characterization of the YmcB and YqeV tRNA methylthiotransferases of Bacillus subtilis.
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 (-SCH(3)). Members of two of the four known subgroups of MTTases have been characterized, typified by MiaB, which modifies N(6)-isopentenyladenosine (i(6)A) to 2-methylthio-N(6)-isopentenyladenosine (ms(2)i(6)A) 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 ms(2)i(6)A formation (MiaB activity), and yqeV is required for modification of N(6)-threonylcarbamoyladenosine (t(6)A) to 2-methylthio-N(6)-threonylcarbamoyladenosine (ms(2)t(6)A) 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 i(6)A from t(6)A 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.
Anton
Brian P
BP
New England Biolabs, Ipswich, MA 01938, USA. anton@neb.com
Russell
Susan P
SP
Vertrees
Jason
J
Kasif
Simon
S
Raleigh
Elisabeth A
EA
Limbach
Patrick A
PA
Roberts
Richard J
RJ
D000241:Adenosine / Q000031:analogs & derivatives; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D017434:Protein Structure, Tertiary; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013466:Sulfurtransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D006360:Heat-Shock Proteins; D011993:Recombinant Fusion Proteins; C104238:yqeV protein, Bacillus subtilis; D012343:RNA, Transfer; D013466:Sulfurtransferases; D000241:Adenosine
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