α-d-(1 → 3)-graft-(1 → 6)-glucan: Comb-like polysaccharide synthesized in vitro with α-1,3/1,6-glucosyltransferase L from Streptococcus salivarius.

We demonstrated that a unique polysaccharide with extremely high molecular weight can be easily obtained via a low-cost, mild reaction in a water medium from sucrose, a photosynthetic product. α-1,3/1,6-Glucosyltransferase L (GtfL) from Streptococcus salivarius produced water-insoluble α-d-glucan from sucrose at 37 °C. Gel permeation chromatography revealed the molecular weight was extremely high; the weight-average molecular weight values were more than 1,000,000 irrespective of the substrate concentration. The Smith degradation of neat glucan and NMR spectroscopic analyses of the acetyl derivative revealed a structure similar to that of a comb-type graft copolymer, α-d-(1 â†’ 3)-graft-(1 â†’ 6)-glucan. The anhydroglucose units (AGUs) in the main-chain backbone are linked by (1 â†’ 3)-glycosidic bonds, whereas a side chain consisting of four AGUs via (1 â†’ 6)-glycosidic bonds alternately extends from C6 of the main chain.

Biochemical and structural characterization of oxygen-sensitive 2-thiouridine synthesis catalyzed by an iron-sulfur protein TtuA.

Two-thiouridine (s2U) at position 54 of transfer RNA (tRNA) is a posttranscriptional modification that enables thermophilic bacteria to survive in high-temperature environments. s2U is produced by the combined action of two proteins, 2-thiouridine synthetase TtuA and 2-thiouridine synthesis sulfur carrier protein TtuB, which act as a sulfur (S) transfer enzyme and a ubiquitin-like S donor, respectively. Despite the accumulation of biochemical data in vivo, the enzymatic activity by TtuA/TtuB has rarely been observed in vitro, which has hindered examination of the molecular mechanism of S transfer. Here we demonstrate by spectroscopic, biochemical, and crystal structure analyses that TtuA requires oxygen-labile [4Fe-4S]-type iron (Fe)-S clusters for its enzymatic activity, which explains the previously observed inactivation of this enzyme in vitro. The [4Fe-4S] cluster was coordinated by three highly conserved cysteine residues, and one of the Fe atoms was exposed to the active site. Furthermore, the crystal structure of the TtuA-TtuB complex was determined at a resolution of 2.5 Å, which clearly shows the S transfer of TtuB to tRNA using its C-terminal thiocarboxylate group. The active site of TtuA is connected to the outside by two channels, one occupied by TtuB and the other used for tRNA binding. Based on these observations, we propose a molecular mechanism of S transfer by TtuA using the ubiquitin-like S donor and the [4Fe-4S] cluster.

Identification of a rhodanese-like protein involved in thiouridine biosynthesis in Thermus thermophilus tRNA.

Incorporation of a sulfur atom into 2-thioribothymidine (s2 T 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 s2 T54 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 s2 T and other sulfur-containing compounds, TtuD has a role to direct sulfur flow to s2 T synthesis.

Folate-/FAD-dependent tRNA methyltransferase from Thermus thermophilus regulates other modifications in tRNA at low temperatures.

TrmFO is a N(5) , N(10) -methylenetetrahydrofolate (CH2 THF)-/FAD-dependent tRNA methyltransferase, which synthesizes 5-methyluridine at position 54 (m(5) U54) in tRNA. Thermus thermophilus is an extreme-thermophilic eubacterium, which grows in a wide range of temperatures (50-83 °C). In T. thermophilus, modified nucleosides in tRNA and modification enzymes form a network, in which one modification regulates the degrees of other modifications and controls the flexibility of tRNA. To clarify the role of m(5) U54 and TrmFO in the network, we constructed the trmFO gene disruptant (∆trmFO) strain of T. thermophilus. Although this strain did not show any growth retardation at 70 °C, it showed a slow-growth phenotype at 50 °C. Nucleoside analysis showed increase in 2'-O-methylguanosine at position 18 and decrease in N(1) -methyladenosine at position 58 in the tRNA mixture from the ∆trmFO strain at 50 °C. These in vivo results were reproduced by in vitro experiments with purified enzymes. Thus, we concluded that the m(5) U54 modification have effects on the other modifications in tRNA through the network at 50 °C. (35) S incorporations into proteins showed that the protein synthesis activity of ∆trmFO strain was inferior to the wild-type strain at 50 °C, suggesting that the growth delay at 50 °C was caused by the inferior protein synthesis activity.

Common thiolation mechanism in the biosynthesis of tRNA thiouridine and sulphur-containing cofactors.

2-Thioribothymidine (s(2)T), a modified uridine, is found at position 54 in transfer RNAs (tRNAs) from several thermophiles; s(2)T stabilizes the L-shaped structure of tRNA and is essential for growth at higher temperatures. Here, we identified an ATPase (tRNA-two-thiouridine C, TtuC) required for the 2-thiolation of s(2)T in Thermus thermophilus and examined in vitro s(2)T formation by TtuC and previously identified s(2)T-biosynthetic proteins (TtuA, TtuB, and cysteine desulphurases). The C-terminal glycine of TtuB is first activated as an acyl-adenylate by TtuC and then thiocarboxylated by cysteine desulphurases. The sulphur atom of thiocarboxylated TtuB is transferred to tRNA by TtuA. In a ttuC mutant of T. thermophilus, not only s(2)T, but also molybdenum cofactor and thiamin were not synthesized, suggesting that TtuC is shared among these biosynthetic pathways. Furthermore, we found that a TtuB-TtuC thioester was formed in vitro, which was similar to the ubiquitin-E1 thioester, a key intermediate in the ubiquitin system. The results are discussed in relation to the mechanism and evolution of the eukaryotic ubiquitin system.