Nonredox thiolation in tRNA occurring via sulfur activation by a [4Fe-4S] cluster

Abstract

Sulfur is present in several nucleosides within tRNAs. In particular, thiolation of the universally conserved methyl-uridine at position 54 stabilizes tRNAs from thermophilic bacteria and hyperthermophilic archaea and is required for growth at high temperature. The simple nonredox substitution of the C2-uridine carbonyl oxygen by sulfur is catalyzed by tRNA thiouridine synthetases called TtuA. Spectroscopic, enzymatic, and structural studies indicate that TtuA carries a catalytically essential [4Fe-4S] cluster and requires ATP for activity. A series of crystal structures shows that (i) the cluster is ligated by only three cysteines that are fully conserved, allowing the fourth unique iron to bind a small ligand, such as exogenous sulfide, and (ii) the ATP binding site, localized thanks to a proteinbound AMP molecule, a reaction product, is adjacent to the cluster. A mechanism for tRNA sulfuration is suggested, in which the unique iron of the catalytic cluster serves to bind exogenous sulfide, thus acting as a sulfur carrier.T he cellular translation machinery contains essential components such as tRNAs. To achieve their function, they feature a great variety of well-conserved posttranscriptional chemical modifications. Sulfur is present in several of these modified nucleosides: thiouridine and derivatives (s 4 U8, s 2 U34, and m 5 s 2 U54), 2-thioadenosine derivatives (ms 2 i 6 A37 and ms 2 t 6 A37), and 2-thiocytidine (s 2 C32). However, mechanisms of sulfur insertion into tRNAs are largely unknown, and the enzymes responsible for these reactions are incompletely characterized. Whereas redox conversion of a C-H to a C-S bond (synthesis of ms 2 i 6 A37 and ms 2 t 6 A37) depends on redox enzymes from the Radical-S-adenosyl-L-methionine iron-sulfur enzyme family, simple nonredox conversion of C = O to C = S group (synthesis of s 2 U34 and s 4 U8) is not expected to require such redox clusters. Intriguingly, we recently discovered that the ATPdependent formation of s 2 C32 in some tRNAs is catalyzed by an iron-sulfur enzyme, TtcA (1). However, the role of its cluster has not been defined. In the same superfamily, TtuA enzymes catalyze the C2-thiolation of uridine 54 in the T loop of thermophilic tRNAs (Fig. 1A), allowing stabilization of tRNAs at high temperature in thermophilic microorganisms. Sequences analysis shows that they share conserved cysteines and ATP binding motif (Fig. S1). Here, we report a detailed biochemical and structural characterization of TtuA that shows the presence of a [4Fe-4S] cluster essential for activity. The crystal structures of Pyrococcus horikoshii TtuA (PhTtuA) show that the cluster, chelated by only three cysteines, is adjacent to the ATP binding site. The presence of electron density near the fourth iron, nonbonded to the protein, indicates that the cluster can bind an exogenous substrate. We propose that thiolation occurs via sulfur binding to the cluster and transfer to the tRNA substrate. The fact that the catalytic [4Fe-4S] cluster serves as a sulfur carrier during a nonredox ...

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

“…6a-c) is considered to be more reasonable because of the following points: first, the sulfur donor protein TtuB is absent in some organisms, including T. maritima and P. horikoshii; second, even TtuA that has a native-partner TtuB (e.g., T. thermophilus TtuA) can transfer a sulfide ion to tRNA; and third, an extra electron density (to which a sulfide ion fits well) was found on the unique Fe site of the [4Fe-4S] cluster of P. horikoshii TtuA (ref. 11 ).…”

“…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).…”

“…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 tRNA (ref. 11 ). In their proposed mechanism, sulfur is first trapped on the unique Fe site of the [4Fe-4S] cluster (hereafter this state is referred to as the [4Fe-5S]-intermediate), from which it is transferred to tRNA.…”

Dependence of heme biosynthesis on iron-sulfur cluster biogenesis: human ALAD is an unrecognized iron-sulfur protein

“…To determine whether human ALAD can indeed coordinate an ISC in human cells, we then overexpressed C-terminally FLAG/MYC-tagged ALAD WT and Mut AFR-AAA in Expi293 cells and puri ed the recombinant proteins anaerobically. Anaerobically puri ed recombinant ALAD migrated as a single band on SDS-PAGE ( Figure 1E) and exhibited a shoulder at ~420 nm in its UV-vis spectrum ( Figure 1F), suggesting the presence of [Fe 4 S 4 ] cluster(s) 32,33,34 .…”