Transfer RNA Bound to MnmH Protein Is Enriched with Geranylated tRNA--A Possible Intermediate in Its Selenation?

PLoS One. 2016 Apr 13;11(4):e0153488. doi: 10.1371/journal.pone.0153488. eCollection 2016.

Abstract

The wobble nucleoside 5-methylaminomethyl-2-thio-uridine (mnm5s2U) is present in bacterial tRNAs specific for Lys and Glu and 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U) in tRNA specific for Gln. The sulfur of (c)mnm5s2U may be exchanged by selenium (Se)-a reaction catalyzed by the selenophosphate-dependent tRNA 2-selenouridine synthase encoded by the mnmH (ybbB, selU, sufY) gene. The MnmH protein has a rhodanese domain containing one catalytic Cys (C97) and a P-loop domain containing a Walker A motif, which is a potential nucleotide binding site. We have earlier isolated a mutant of Salmonella enterica, serovar Typhimurium with an alteration in the rhodanese domain of the MnmH protein (G67E) mediating the formation of modified nucleosides having a geranyl (ge)-group (C10H17-fragment) attached to the s2 group of mnm5s2U and of cmnm5s2U in tRNA. To further characterize the structural requirements to increase the geranylation activity, we here report the analysis of 39 independently isolated mutants catalyzing the formation of mnm5ges2U. All these mutants have amino acid substitutions in the rhodanese domain demonstrating that this domain is pivotal to increase the geranylation activity. The wild type form of MnmH+ also possesses geranyltransferase activity in vitro although only a small amount of the geranyl derivatives of (c)mnm5s2U is detected in vivo. The selenation activity in vivo has an absolute requirement for the catalytic Cys97 in the rhodanese domain whereas the geranylation activity does not. Clearly, MnmH has two distinct enzymatic activities for which the rhodanese domain is pivotal. An intact Walker motif in the P-loop domain is required for the geranylation activity implying that it is the binding site for geranylpyrophosphate (GePP), which is the donor molecule in vitro in the geranyltransfer reaction. Purified MnmH from wild type and from the MnmH(G67E) mutant have bound tRNA, which is enriched with geranylated tRNA. This in conjunction with earlier published data, suggests that this bound geranylated tRNA may be an intermediate in the selenation of the tRNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Escherichia coli / genetics
  • Phosphates / metabolism
  • RNA, Bacterial / genetics
  • RNA, Bacterial / metabolism*
  • RNA, Transfer / genetics
  • RNA, Transfer / metabolism*
  • Salmonella typhimurium / genetics
  • Selenium / metabolism*
  • Selenium Compounds / metabolism
  • Sulfurtransferases / genetics
  • Sulfurtransferases / metabolism*
  • Thiosulfate Sulfurtransferase / genetics
  • Thiosulfate Sulfurtransferase / metabolism

Substances

  • Phosphates
  • RNA, Bacterial
  • Selenium Compounds
  • selenophosphate
  • RNA, Transfer
  • Sulfurtransferases
  • tRNA 2-selenouridine synthase
  • Thiosulfate Sulfurtransferase
  • Selenium

Grants and funding

This work was supported by grants from the Swedish Science Research Council (BU-2930) and from the Carl Trygger Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.