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

Abstract

Two-thiouridine (s 2 U) at position 54 of transfer RNA (tRNA) is a posttranscriptional modification that enables thermophilic bacteria to survive in high-temperature environments. s 2 U 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.Fe-S cluster | sulfur transfer | tRNA modification | 2-thiouridine | crystal structure

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

“…2a). The manner of interactions between TtuA and Fe-S (i.e., TtuA coordinately bound to Fe-S via three cysteine residues) was identical to that for holo-TtuA reported previously 10 . However, a new interaction directly coordinated between the unique Fe site of the [4Fe-4S] cluster and the C-terminus of TtuB was observed (Fig.…”

“…Examples include: TtuA, which catalyzes the formation of 2-thioribothymidin (s 2 T) at position 54 (refs. 10,11 ); TtcA, which catalyzes the formation of 2-thiocytidine (s 2 C) at position 32 (ref. 12 ); and ThiI, which catalyzes the formation of 4-thiouridine (s 4 U) at position 8 (ref.…”

“…TtuA is the best-characterized enzyme among the Fe-Sdependent sulfur transferases 10,11,[14][15][16] and is known to contain a [4Fe-4S] type cluster in the catalytic site ( Fig. 1).…”

Enzymatic Thioamide Formation in a Bacterial Antimetabolite Pathway

“…Since the timing of thioamide formation in the 6TG pathway was unknown, all enzyme assays were performed in parallel with an umber of potential precursors:g uanine (2), guanosine (3), guanosine monophosphate (GMP, 4), guanosine diphosphate (GDP, 5), and guanosine triphosphate (GTP, 6;F igure 2A). Since inorganic sulfide has previously been shown to function as as ulfur source for AANH enzymes in vitro, [9] we added lithium sulfide to the reaction mixtures and product formation was monitored by HPLC.H owever, none of the substrates were converted into the corresponding thioamide;arepresentative trace with 4 is displayed in Figure 2B.…”

“…Although product peaks were not observed in the HPLC profiles of reactions with guanine and guanosine,n ew peaks with aU Vabsorbance at 340 nm, which is characteristic for 6TG,w ere formed in assays with each of the three tested guanine nucleotides ( Figure 2F). By comparison with synthetic references (Figure S4), we identified these new compounds as the corresponding thioamides, 6-thio GMP (6TGMP, 8, Figure 2G), 6-thio GDP (6TGDP, 9), and 6-thio GTP (6TGTP, 10). Additionally,b yu sing LC-HMRS we unequivocally confirmed that the new compounds are the expected thioamides.D ata for the formation of 8 is presented in Figures S5-6.…”