The [4Fe-4S] cluster of sulfurtransferase TtuA desulfurizes TtuB during tRNA modification in Thermus thermophilus

Minghao Chen,Min Yao,Yoshikazu Tanaka,Masato Ishizaka,Masaki Horitani,Naoki Shigi,Shun Narai

doi:10.1038/s42003-020-0895-3

Abstract

TtuA and TtuB are the sulfurtransferase and sulfur donor proteins, respectively, for biosynthesis of 2-thioribothymidine (s2T) 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.

Highlights

TtuA and TtuB are the sulfurtransferase and sulfur donor proteins, respectively, for biosynthesis of 2-thioribothymidine (s2T) at position 54 of transfer RNA, which is responsible for adaptation to high temperature environments in Thermus thermophilus
The atom was not identified but, because the electron density was more consistent with a hydrosulfide rather than a hydroxide ion, Arragain et al proposed that the unique Fe site of TtuA is responsible for capturing the sulfide ion and transferring it to the substrate transfer RNA (tRNA)
The biosynthesis of s2T is composed of two sequential reactions: adenylation (Fig. 6e, f) and thiolation (Fig. 6f, g)

Summary

Introduction

TtuA and TtuB are the sulfurtransferase and sulfur donor proteins, respectively, for biosynthesis of 2-thioribothymidine (s2T) at position 54 of transfer RNA (tRNA), which is responsible for adaptation to high temperature environments in Thermus thermophilus. 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. It functions as an intrinsic proteinous sulfur donor in the TtuA reaction[14], supplying a sulfur atom as a thiocarboxylate moiety at its C-terminus, TtuB-COSH Similar usage of a thiocarboxylate moiety to protect and transport a sulfur atom is known for the biosynthetic pathways of both thiamin[19] and molybdenum cofactor[20] For both pathways, the sulfur atom is transferred directly from the donor protein to the substrate, so an Fe-S cluster is not required

Methods

Results

Conclusion