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

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.

S-Geranyl-2-thiouridine wobble nucleosides of bacterial tRNAs; chemical and enzymatic synthesis ofS-geranylated-RNAs and their physicochemical characterization

“…As expected, we found that a single geranyl modification increases the hydrophobicity of the geS2U–RNA strand in comparison to the reference U (ΔLog P = 0.27) and S (ΔLog P = 0.10) RNA strands (Table 2). Although it has been suggested that the presence of a hydrophobic geranyl residue in the tRNAs affects their intracellular localization and transport through the cell membranes (11,15), recent studies have shown a lack of geranylated tRNA enrichment in outer or inner cellular membranes (9). …”

“…In earlier studies, we chemically transformed S2U to Se2U via S -geranyl-2-thiouridine and suggested a possible linear mechanism for the SelU-catalyzed reaction, in which geS2U exists as an intermediate in the cellular transformation of S2U to Se2U (Figure 5) (14). Such a possibility has been recently considered in advanced studies on tRNA selenouridine synthase (9). …”

“…These modifications are essential for the viability of eukaryotic and prokaryotic cells. The absence of a side chain in the C5 position or the lack of a sulfur atom in the C2 position leads to detrimental frame-shift reading (9). …”

“…The bacterial tRNA selenouridine synthase (SelU) has been recognized not only as the biocatalyst for the S -geranylation of tRNAs in cells (9–11) but also as the catalyst for S→Se replacement (13). Liu et al.…”

“…This hypothesis was supported significantly by our success in performing the two-step chemical transformation S2U→geS2U→Se2U and was further supported by evidence from Jager et al. (9). Several mutations introduced into the SelU (MnmH) polypeptide chain allowed the amino acids responsible for interactions within the tRNA molecule and for binding of modified nucleosides (the Walker's motif in the P-loop domain) to be identified.…”