Naoki Shigi

Ubiquitin-like proteins (Ubls) in eukaryotes and bacteria mediate sulfur transfer for the biosynthesis of sulfur-containing biomolecules and form conjugates with specific protein targets to regulate their functions. Here, we investigated the functions and physiological importance of Ubls in a hyperthermophilic archaeon by constructing a series of deletion mutants. We found that the Ubls (TK1065, TK1093, and TK2118) in Thermococcus kodakarensis are conjugated to their specific target proteins, and all three are involved in varying degrees in the biosynthesis of sulfur-containing biomolecules such as tungsten cofactor (Wco) and tRNA thiouridines. TK2118 (named UblB) is involved in the biosynthesis of Wco in a glyceraldehyde 3-phosphate:ferredoxin oxidoreductase, which is required for glycolytic growth, whereas TK1093 (named UblA) plays a key role in the efficient thiolation of tRNAs, which contributes to cellular thermotolerance. Intriguingly, in the presence of elemental sulfur (S ⁰ ) in the culture medium, defective synthesis of these sulfur-containing molecules in Ubl mutants was restored, indicating that T. kodakarensis can use S ⁰ as an alternative sulfur source without Ubls. Our analysis indicates that the Ubl-mediated sulfur-transfer system in T. kodakarensis is important for efficient sulfur assimilation, especially under low S ⁰ conditions, which may allow this organism to survive in a low sulfur environment. IMPORTANCE: Sulfur is a crucial element in living organisms, occurring in various sulfur-containing biomolecules including iron-sulfur clusters, vitamins, and RNA thionucleosides, as well as the amino acids cysteine and methionine. In archaea, the biosynthesis routes and sulfur donors of sulfur-containing biomolecules are largely unknown. Here, we explored the functions of Ubls in the deep-blanched hyperthermophilic archaeon, Thermococcus kodakarensis . We demonstrated functional redundancy of these proteins in the biosynthesis of tungsten cofactor and tRNA thiouridines and the significance of these sulfur-carrier functions, especially in low sulfur environments. We propose that acquisition of a Ubl sulfur-transfer system, in addition to an ancient inorganic sulfur assimilation pathway, enabled the primordial archaeon to advance into lower-sulfur environments and expand their habitable zone.

4-Thiouridine (s⁴U) is a modified nucleoside, found at positions 8 and 9 in tRNA from eubacteria and archaea. Studies of the biosynthetic pathway and physiological role of s⁴U in tRNA are ongoing in the tRNA modification field. s⁴U has also recently been utilized as a biotechnological tool for analysis of RNAs. Therefore, a selective and sensitive system for the detection of s⁴U is essential for progress in the fields of RNA technologies and tRNA modification. Here, we report the use of biotin-coupled 2-aminoethyl-methanethiosulfonate (MTSEA biotin-XX) for labeling of s⁴U and demonstrate that the system is sensitive and quantitative. This technique can be used without denaturation; however, addition of a denaturation step improves the limit of detection.Thermus thermophilustRNAs, which abundantly contain 5-methyl-2-thiouridine, were tested to investigate the selectivity of the MTSEA biotin-XX s⁴U detection system. The system did not react with 5-methyl-2-thiouridine in tRNAs from aT. thermophilustRNA 4-thiouridine synthetase (thiI) gene deletion strain. Thus, the most useful advantage of the MTSEA biotin-XX s⁴U detection system is that MTSEA biotin-XX reacts only with s⁴U and not with other sulfur-containing modified nucleosides such as s²U derivatives in tRNAs. Furthermore, the MTSEA biotin-XX s⁴U detection system can analyze multiple samples in a short time span. The MTSEA biotin-XX s⁴U detection system can also be used for the analysis of s⁴U formation in tRNA. Finally, we demonstrate that the MTSEA biotin-XX system can be used to visualize newly transcribed tRNAs inS. cerevisiaecells.

Various sulfur-containing biomolecules include iron–sulfur clusters that act as cofactors for enzymes, sulfur-containing vitamins such as thiamin, and sulfur-modified nucleosides in RNA, in addition to methionine and cysteine in proteins. Sulfur-containing nucleosides are post-transcriptionally introduced into tRNA molecules, where they ensure precise codon recognition or stabilization of tRNA structure, thereby maintaining cellular proteome integrity. Modulating sulfur modification controls the translation efficiency of specific groups of genes, allowing organisms to adapt to specific environments. The biosynthesis of tRNA sulfur nucleosides involves elaborate ‘sulfur trafficking systems’ within cellular sulfur metabolism and ‘modification enzymes’ that incorporate sulfur atoms into tRNA. This review provides an up-to-date overview of advances in our knowledge of the mechanisms involved. It covers the functions, biosynthesis, and biodegradation of sulfur-containing nucleosides as well as the reaction mechanisms of biosynthetic enzymes catalyzed by the iron–sulfur clusters, and identification of enzymes involved in the de-modification of sulfur atoms of RNA. The mechanistic similarity of these opposite reactions is discussed. Mutations in genes related to these pathways can cause human diseases (e.g., cancer, diabetes, and mitochondrial diseases), emphasizing the importance of these pathways.

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.

Transfer RNA (tRNA) is an adaptor molecule indispensable for assigning amino acids to codons on mRNA during protein synthesis. 2-thiouridine (s²U) derivatives in the anticodons (position 34) of tRNAs for glutamate, glutamine, and lysine are post-transcriptional modifications essential for precise and efficient codon recognition in all organisms. s²U34 is introduced either by (i) bacterial MnmA/eukaryote mitochondrial Mtu1 or (ii) eukaryote cytosolic Ncs6/archaeal NcsA, and the latter enzymes possess iron-sulfur (Fe–S) cluster. Here, we report the identification of novel-type MnmA homologs containing three conserved Cys residues, which could support Fe–S cluster binding and catalysis, in a broad range of bacteria, including thermophiles, Cyanobacteria, Mycobacteria, Actinomyces, Clostridium, and Helicobacter. Using EPR spectroscopy, we revealed that Thermus thermophilus MnmA (TtMnmA) contains an oxygen-sensitive [4Fe–4S]-type cluster. Efficient in vitro formation of s²U34 in tRNA^Lys and tRNA^Gln by holo-TtMnmA occurred only under anaerobic conditions. Mutational analysis of TtMnmA suggested that the Fe–S cluster is coordinated by the three conserved Cys residues (Cys105, Cys108, and Cys200), and is essential for its activity. Evolutionary scenarios for the sulfurtransferases, including the Fe–S cluster containing Ncs6/NcsA s²U thiouridylases and several distantly related sulfurtransferases, are proposed.

Sulfur is an essential element in all living organisms. In tRNA molecules, there are many sulfur-containing nucleosides, introduced post-transcriptionally, that function to ensure proper codon recognition or stabilization of tRNA structure, thereby enabling accurate and efficient translation. The biosynthesis of tRNA sulfur modifications involves unique sulfur trafficking systems that are closely related to cellular sulfur metabolism, and ‘modification enzymes’ that incorporate sulfur atoms into tRNA. Herein, recent biochemical and structural characterization of the biosynthesis of sulfur modifications in tRNA is reviewed, with special emphasis on the reaction mechanisms of modification enzymes. It was recently revealed that TtuA/Ncs6-type 2-thiouridylases from thermophilic bacteria/archaea/eukaryotes are oxygen-sensitive iron-sulfur proteins that utilize a quite different mechanism from other 2-thiouridylase subtypes lacking iron-sulfur clusters such as bacterial MnmA. The various reaction mechanisms of RNA sulfurtransferases are also discussed, including tRNA methylthiotransferase MiaB (a radical S-adenosylmethionine-type iron-sulfur enzyme) and other sulfurtransferases involved in both primary and secondary sulfur-containing metabolites.

Significance: One of the posttranscriptional modifications of tRNA, 2-thiouridine (s ² U), enhances thermostability. Although extensive studies have been conducted to understand the mechanism behind this modification, many ill-defined points remain, because the S-transfer enzyme 2-thiouridine synthetase TtuA has shown very low activity in previous in vitro experiments. Here we demonstrate that TtuA requires oxygen-labile [4Fe-4S] clusters for its activity. Furthermore, we determine the crystal structure of TtuA in complex with the Fe-S cluster and ATP analog and also with its S-donor protein, 2-thiouridine synthesis sulfur carrier protein (TtuB). The combined actions of TtuA and TtuB using the Fe-S cluster aid the S-transfer mechanism.

Incorporation of a sulfur atom into 2‐thioribothymidine (s²T or 5‐methyl‐2‐thiouridine) at position 54 in thermophile tRNA is accomplished by an elaborate system composed of many proteins which confers thermostability to the translation system. We identified ttuD (tRNA‐two‐thiouridine D) as a gene for the synthesis of s²T54 in Thermus thermophilus. The rhodanese‐like protein TtuD enhances the activity of cysteine desulfurases and receives the persulfide generated by cysteine desulfurases in vitro. TtuD also enhances the formation of thiocarboxylated TtuB, the sulfur donor for the tRNA sulfurtransferase TtuA. Since cysteine desulfurases are the first enzymes in the synthesis of s²T and other sulfur‐containing compounds, TtuD has a role to direct sulfur flow to s²T synthesis.

The ubiquitin-like protein TtuB is a sulfur carrier for the biosynthesis of 2-thioribothymidine (s²T) at position 54 in some thermophilic bacterial tRNAs. TtuB captures a S atom at its C-terminus as a thiocarboxylate and transfers it to tRNA by the transferase activity of TtuA. TtuB also functions to suppress s²T formation by forming a covalent bond with TtuA. To explore how TtuB interacts with TtuA and switches between these two different functions, high-resolution structure analysis of the TtuA–TtuB complex is required. In this study, the TtuA–TtuB complex fromThermus thermophiluswas expressed, purified and crystallized. To mimic the thiocarboxylated TtuB, the C-terminal Gly residue was replaced with Cys (G65C) to obtain crystals of the TtuA–TtuB complex. A Zn-MAD data set was collected to a resolution of 2.5 Å. MAD analysis successfully determined eight Zn sites, and a partial structure model composed of four TtuA–TtuB complexes in the asymmetric unit was constructed.