Structure-based mechanistic insights into catalysis by tRNA thiolation enzymes

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Highlights

  • tRNA thiolation enzymes were thought to use a persulfide on a catalytic cysteine as a sulfur source.

  • Several tRNA thiolation enzymes contain a [4Fe–4S] cluster acting as a sulfur carrier.

  • Previously studied enzymes with unexplained mechanism should be reexamined.

  • Production under anaerobic conditions and cluster reconstitution should be tested.

In all domains of life, ribonucleic acid (RNA) maturation includes post-transcriptional chemical modifications of nucleosides. Many sulfur-containing nucleosides have been identified in transfer RNAs (tRNAs), such as the derivatives of 2-thiouridine (s2U), 4-thiouridine (s4U), 2-thiocytidine (s2C), 2-methylthioadenosine (ms2A). These modifications are essential for accurate and efficient translation of the genetic code from messenger RNA (mRNA) for protein synthesis. This review summarizes the recent discoveries concerning the mechanistic and structural characterization of tRNA thiolation enzymes that catalyze the non-redox substitution of oxygen for sulfur in nucleosides. Two mechanisms have been described. One involves persulfide formation on catalytic cysteines, while the other uses a [4Fe–4S] cluster, chelated by three conserved cysteines only, as a sulfur carrier.

Introduction

tRNAs are key players in genetic code decoding, a fundamental process in all living organisms. All tRNAs feature post-transcriptional chemical modifications [1] that stabilize their tertiary structure and fine-tune the decoding process [2,3]. Sulfur, an essential element in life, is present in several cofactors and tRNAs: at positions 8, 9 in the core, 32, 33, 34, 37 around the anticodon and 54 in the T-loop (Figure 1a) [4,5]. The formation of 2-thiouridine (s2U), 4-thiouridine (s4U), 2-thiocytidine (s2C) and 2-methylthioadenosine (ms2A) is catalyzed by specific enzymes called ThiI, TtcA, MnmA/Ctu1/Tuc1/Ncs6, MiaB and MtaB, TtuA [4,5] acting at positions 8, 32, 34, 37 and 54, respectively (Figure 1a). Because most thiI genes play no role in thiamine biosynthesis [6], ThiI is renamed here TtuI for tRNA thiouridine I.

There are two main classes of tRNA thiolation reactions. The insertion of sulfur within an inert Csingle bondH bond is an [Fe–S]-dependent redox reaction catalyzed by the radical S-adenosyl-l-methionine (SAM) methylthiotransferases MiaB and MtaB. Because their structures remain unknown and their mechanisms have recently been reviewed [7,8], this class will not be discussed here. The non-redox substitution of oxygen for sulfur (Figure 1a) is catalyzed by ATP-dependent tRNA thiolases that share a pyrophosphatase (PPase) domain (Figure S1). We review here their crystal structures and catalytic mechanisms in light of research from the last two years showing that several of these enzymes are dependent on a [4Fe–4S] cluster (Table S1).

Section snippets

Formation of persulfides on reactive cysteines

The biosynthesis of sulfur-containing nucleosides involves several proteins that relay sulfur atoms originating from l-cysteine to tRNA [4,9,10]. In most cases, a pyridoxal-5′-phosphate-dependent cysteine desulfurase (IscS/Nsf1, YrvO, Nifz) first uses l-cysteine to form an enzyme-bound cysteine persulfide whose sulfur is next transferred to an acceptor protein [11, 12, 13, 14, 15]. This transfer is usually monitored by detecting, upon incubation with [35S]-l-cysteine, radioactive sulfur on the

U8-tRNA 4-sulfurtransferase TtuI (tRNA thiouridine I)

s4U at position 8 in the loop connecting the acceptor and D-stems of bacterial and archaeal tRNAs (Figure 1a) mediates cellular responses to UV stress [32]. In E. coli and Bacillus subtilis, TtuI and the cysteine desulfurase IscS [33,34] or NifZ [13], respectively, are required for s4U8-tRNA thiolation. TtuI enzymes have three conserved domains (Figures S1 and 2 a). Genomic analysis of the ttuI gene family identified two groups [9]: organisms like E. coli [35] that possess an additional

C32-tRNA 2-sulfurtransferase TtcA (tRNA-2-thiocytidine A)

TtcA enzymes target cytidine at position 32 near the anticodon in tRNAs (Figure 1a). The [Fe–S]-dependent TtcA/TtuA family was first identified following the characterization of E. coli and Salmonella thyphimurium strains deficient in s2C-modified tRNAs [44]. This class is characterized by a CXXC sequence motif in the central region (Figure S1). Analysis of tRNA from mutated strains indicated that the two cysteines in this motif are required for s2C formation [44] . Site-directed mutagenesis

Thiolation of U34

Sulfuration of U34 at the wobble position of the anticodon in Glu-tRNA, Gln-tRNA and Lys-tRNA (Figure 1a) is conserved in all organisms and guarantees fidelity of protein translation [46]. Lack of s2U34-tRNA results in severe growth reduction [12,15,18,47, 48, 49]. Two distinct enzyme families of the MnmA-types and Ncs6-types catalyze s2U34-tRNA formation (Figure S1). MnmA-like proteins operate in bacteria [22,50,51] and mitochondria [52], and Ncs6-like proteins in archaea and the eukaryotic

U54-tRNA 2-sulfurtransferase TtuA (tRNA-2-thiouridine A)

s2U at position 54 in the T-loop of tRNAs (Figure 1a) stabilizes its ternary structure in thermophilic bacteria and archaea for growth at high temperature [25]. Spectroscopic and biochemical analyses have shown that TtTtuA, PhTtuA and TtuA from T. maritima use a [4Fe–4S] cluster for U54-tRNA thiolation [28••,55••]. Thiolation did not occur in the absence of a sulfur source (Na2S [28••,55••] or TtTtuB-COSH [28••]), indicating that the sulfur atom incorporated into the nucleoside does not come

Conclusion

Although a general mechanism for tRNA thiolation was initially proposed, in which a persulfide attached to a catalytic cysteine is the sulfur donor for tRNA thiolation [16,51,57], there is increasing evidence that a sulfur-containing species bound to a [4Fe–4S] cluster, ligated to three cysteines only, can be the sulfurating agent [28••,31••,55••, Bimai, unpublished]. According to this finding, the tRNA thiolation enzymes for which a low in vitro activity has been detected and/or for which the

Conflict of interest statement

Nothing declared.

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

We thank Marc Fontecave for sharing his enthusiasm for the iron–sulfur field and giving us competent advices all along this work.

This work was supported by the French State Program ‘Investissements d’Avenir’ (Grants ‘LABEX DYNAMO’, ANR-11-LABX-0011) and the CNRS.

References (57)

  • N. Shigi

    Posttranslational modification of cellular proteins by a ubiquitin-like protein in bacteria

    J Biol Chem

    (2012)
  • E.G. Mueller et al.

    The role of the cysteine residues of ThiI in the generation of 4-thiouridine in tRNA

    J Biol Chem

    (2001)
  • C.T. Lauhon et al.

    The iscS gene in Escherichia coli is required for the biosynthesis of 4-thiouridine, thiamin, and NAD

    J Biol Chem

    (2000)
  • D. You et al.

    Direct evidence that ThiI is an ATP pyrophosphatase for the adenylation of uridine in 4-thiouridine biosynthesis

    ChemBioChem

    (2008)
  • C.M. Wright et al.

    A paradigm for biological sulfur transfers via persulfide groups: a persulfide-disulfide-thiol cycle in 4-thiouridine biosynthesis

    Chem Commun

    (2002)
  • P. Neumann et al.

    Crystal structure of a 4-thiouridine synthetase-RNA complex reveals specificity of tRNA U8 modification

    Nucleic Acids Res

    (2014)
  • M. Sugahara et al.

    Purification, crystallization and preliminary crystallographic analysis of the putative thiamine-biosynthesis protein PH1313 from Pyrococcus horikoshii OT3

    Acta Crystallogr Sect F Struct Biol Cryst Commun

    (2007)
  • R. Kambampati et al.

    MnmA and IscS are required for in vitro 2-thiouridine biosynthesis in Escherichia coli

    Biochemistry

    (2003)
  • G.R. Björk et al.

    A conserved modified wobble nucleoside (mcm5s2U) in lysyl-tRNA is required for viability in yeast

    RNA

    (2007)
  • P. Boccaletto et al.

    MODOMICS: a database of RNA modification pathways. 2017 update

    Nucleic Acids Res

    (2018)
  • B. El Yacoubi et al.

    Biosynthesis and function of posttranscriptional modifications of transfer RNAs

    Annu Rev Genet

    (2012)
  • N. Shigi

    Biosynthesis and functions of sulfur modifications in tRNA

    Front Genet

    (2014)
  • N. Shigi

    Recent advances in our understanding of the biosynthesis of sulfur modifications in tRNAs

    Front Microbiol

    (2018)
  • R.A. Bender

    The danger of annotation by analogy: most "thiI" genes play no role in thiamine biosynthesis

    J Bacteriol

    (2011)
  • E. Mulliez et al.

    On the role of additional [4Fe-4S] clusters with a free coordination site in radical-SAM enzymes

    Front Chem

    (2017)
  • M. Kotera et al.

    Comprehensive genomic analysis of sulfur-relay pathway genes

    Genome Inform

    (2010)
  • M. Cavuzic et al.

    Biosynthesis of sulfur-containing tRNA modifications: a comparison of bacterial, archaeal, and eukaryotic pathways

    Biomolecules

    (2017)
  • C.T. Lauhon

    Requirement for IscS in biosynthesis of all thionucleosides in Escherichia coli

    J Bacteriol

    (2002)
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    1

    Present address: Department of Biochemistry and Biophysics, Stockholm University, Sweden.

    2

    Present address: IFP Energies nouvelles, 1 et 4 avenue de Bois-Préau, 92852 Rueil-Malmaison, France.

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    Equivalent contribution.

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