3 research outputs found
Quick and Spontaneous Transformation between [3Feā4S] and [4Feā4S] IronāSulfur Clusters in the tRNA-Thiolation Enzyme TtuA
Ironāsulfur (FeāS) clusters are essential cofactors for enzyme activity. These FeāS clusters are present in structurally diverse forms, including [4Feā4S] and [3Feā4S]. Type-identification of the FeāS cluster is indispensable in understanding the catalytic mechanism of enzymes. However, identifying [4Feā4S] and [3Feā4S] clusters in particular is challenging because of their rapid transformation in response to oxidationāreduction events. In this study, we focused on the relationship between the FeāS cluster type and the catalytic activity of a tRNA-thiolation enzyme (TtuA). We reconstituted [4Feā4S]-TtuA, prepared [3Feā4S]-TtuA by oxidizing [4Feā4S]-TtuA under strictly anaerobic conditions, and then observed changes in the FeāS clusters in the samples and the enzymatic activity in the time-course experiments. Electron paramagnetic resonance analysis revealed that [3Feā4S]-TtuA spontaneously transforms into [4Feā4S]-TtuA in minutes to one hour without an additional free Fe source in the solution. Although the TtuA immediately after oxidation of [4Feā4S]-TtuA was inactive [3Feā4S]-TtuA, its activity recovered to a significant level compared to [4Feā4S]-TtuA after one hour, corresponding to an increase of [4Feā4S]-TtuA in the solution. Our findings reveal that [3Feā4S]-TtuA is highly inactive and unstable. Moreover, time-course analysis of structural changes and activity under strictly anaerobic conditions further unraveled the FeāS cluster type used by the tRNA-thiolation enzyme
The [4Fe-4S] cluster of sulfurtransferase TtuA desulfurizes TtuB during tRNA modification in Thermus thermophilus
TtuA and TtuB are the sulfurtransferase and sulfur donor proteins, respectively, for biosynthesis of 2-thioribothymidine (s(2)T) at position 54 of transfer RNA (tRNA), which is responsible for adaptation to high temperature environments in Thermus thermophilus. The enzymatic activity of TtuA requires an iron-sulfur (Fe-S) cluster, by which a sulfur atom supplied by TtuB is transferred to the tRNA substrate. Here, we demonstrate that the Fe-S cluster directly receives sulfur from TtuB through its inherent coordination ability. TtuB forms a [4Fe-4S]-TtuB intermediate, but that sulfur is not immediately released from TtuB. Further desulfurization assays and mutation studies demonstrated that the release of sulfur from the thiocarboxylated C-terminus of TtuB is dependent on adenylation of the substrate tRNA, and the essential residue for TtuB desulfurization was identified. Based on these findings, the molecular mechanism of sulfur transfer from TtuB to Fe-S cluster is proposed. Chen et al. demonstrate how the Fe-S cluster receives sulfur from TtuB, a ubiquitin-like sulfur donor during tRNA modification. They find that the release of sulfur from the thiocarboxylated C-terminus of TtuB depends on the adenylation of the substrate tRNA. This study provides molecular insights into the sulfur modification of tRNA