# The thiolation of U34 at carbon 2 in tRNA by Escherichia coli MnmA precedes modification at carbon 5 and is dependent on a [4Fe-4S] cluster

Sylvain Gervason, Sambuddha Sen, Jingjing Zhou, Karolina Podskoczyj, Grazyna Leszczynska, Sylvain Caillat, Jean-Luc Ravanat, Marc Fontecave, Béatrice Golinelli-Pimpaneau

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Sylvain Gervason, Sambuddha Sen, Jingjing Zhou, Karolina Podskoczyj, Grazyna Leszczynska, et al. The thiolation of U34 at carbon 2 in tRNA by Escherichia coli MnmA precedes modification at carbon 5 and is dependent on a [4Fe-4S] cluster. Journal of Inorganic Biochemistry, 2025, 274, pp.113064. 10.1016/j.jinorgbio.2025.113064. hal-05248485v2

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The thiolation of U34 at carbon 2 in tRNA by *Escherichia coli* MnmA precedes modification at carbon 5 and is dependent on a [4Fe-4S] cluster

Sylvain Gervason<fnref n="1" />, Sambuddha Sen<fnref n="1" />, Jingjing Zhou<fnref n="1" />, Marouane Libiad<fnref n="1" />, Karolina Podskoczyj<fnref n="2" />,
Grazyna Leszczynska<fnref n="2" />, Sylvain Caillat<fnref n="3" />, Jean-Luc Ravanat<fnref n="3" />, Marc Fontecave<fnref n="1" />, Béatrice Golinelli-
Pimpaneau<fnref n="1" />*

**Abstract**

Chemical modifications of transfer RNAs (tRNAs) fine-tune the recognition of messenger RNA codons on the ribosome for accurate protein translation. Uridine 34 (U34) of the anticodon of three tRNAs is modified in all domains of life. In bacteria, U34 is modified at C5 by aminomethyl derivatives by MnmE/MnmG enzymes and at C2 by a sulfur atom by MnmA enzymes from two distinct classes. C-type MnmAs possess a CXXC+ C catalytic motif, in which cysteines bind a [4Fe-4S] cluster essential for catalysis. D-type MnmAs possess a DXXC+ C catalytic motif, which can also bind a [4Fe-4S] cluster, but the function of the cluster is controversial. To shed light on the role of the [4Fe-4S] cluster in the thiolation reaction, we report here new *in vitro* catalytic assays of the D-type *Escherichia coli* MnmA enzyme (EcMnmA) before, and after cluster reconstitution under anaerobic conditions. We confirm that that only holo-EcMnmA can thiolate U34 in a tRNA transcript using sulfide as a sulfur source and show that 1) a bulk *E. coli* Δ*mnmA* tRNA, containing the C5 modification, is not a substrate, indicating that thiolation at C2 precedes modification at C5, 2) cysteine with IscS cannot provide the sulfur atom for tRNA thiolation without a reductant and 3) the variant, in which the aspartate of the DXXC + C motif is replaced by cysteine, is efficient in U34-tRNA thiolation *in vitro* but not *in vivo*, raising questions about the *in vivo* function of soft cluster coordination.

**Keywords**

MnmA, sulfuration, thiolation, thiouridylase, [4Fe-4S] cluster, sulfur transferase

<footnote n="1">¹ Laboratoire de Chimie des Processus Biologiques, UMR 8229 CNRS, Collège de France, Sorbonne Université, 11 Place Marcelin Berthelot, 75231 Paris cedex 05, France</footnote>

<footnote n="2">² Institute of Organic Chemistry, Faculty of Chemistry, Lodz University of Technology, Zeromskiego 116, 90-924 Lodz, Poland</footnote>

<footnote n="3">³ University of Grenoble Alpes, CEA, CNRS, IRIG, SyMMES UMR 5819, F-38000 Grenoble, France
*Corresponding author</footnote>
# 1 Introduction

Post-transcriptional modifications in tRNA ensure the correct folding of tRNA and guarantee the fidelity and efficiency of translation [1, 2], especially those found near the anticodon loop [3, 4]. Recent studies have revealed that tRNA modifications can be dynamically altered in response to levels of cellular metabolites and environmental stresses [5, 6]. Deficiencies in tRNA modification have been shown to be linked to mitochondrial diseases, neurological disorders and cancer [7, 8]. Up to now thionucleosides have been identified only in tRNA, at five different positions [9-11]. In particular uridine at position 34 in the anticodon is modified by a sulfur atom at carbon 2 [12-17] and by aminomethyl derivatives (methylaminomethyl (mnm) or carboxymethylaminomethyl (cmnm) [18-21] at carbon 5 in three tRNAs, tRNA<sup>Gln</sup><sub>UUG</sub>, tRNA<sup>Lys</sup><sub>UUU</sub> and tRNA<sup>Glu</sup><sub>UUC</sub> in all domains of life. Absence of 2-thiolation of U34, or both C2 and C5 modifications of U34 yields to nonviability in several organisms, such as *Bacillus subtilis* [14], yeast [22], *Salmonella enterica serovar typhimurium* [23] and *Escherichia coli* [24]. In *E. coli*, it has been shown that the tRNA thiolation pathway modulates the intracellular redox state [25] and that the 2-thiolation of U34 enables the bacterium to resist to acid stresses and to exposure to H<sub>2</sub>O<sub>2</sub> [16, 24].

The nonredox substitution of an oxygen atom by a sulfur atom in U34-tRNA is catalyzed by two types of ATP-dependent enzymes (Figure 1A) that are distinct both in terms of sequence and structure [11, 26]: MnmA-type enzymes in bacteria [12, 14-16, 27] and mitochondria [28] and NcsA-type enzymes (also called Ncs6 or CTU1) in archaea [17] and the cytosol of eukaryotes [13]. In addition, sequence comparison of bacterial MnmA proteins led to their classification into two subgroups: C-type proteins such as *Thermus thermophilus* MnmA (TtMnmA), that like NcsA-type enzymes possess three conserved cysteines belonging to a CXXC + C motif at their catalytic site and D-type proteins such as MnmA from *E. coli* (EcMnmA) that possess a DXXC + C motif with an aspartate replacing the first cysteine in the catalytic motif [15]. Spectroscopic and mutagenesis studies, together with catalytic assays using inorganic sulfide as a sulfur source, have shown that the three cysteines of the conserved motif of NcsA from *Methanococcus maripaludis* (MmNcsA) [17] and TtMnmA [15] bind a [4Fe-4S] cluster that is essential for catalysis, as is the case for several other tRNA thiolation enzymes [26]. Such a coordination of the [4Fe-4S] cluster was supported by the crystal structure of MmNcsA [17] and by the AlphaFold2 model of TtMnmA [26].

The case of D-type MnmA proteins is a matter of controversy [12, 16, 29, 30]. Based on the crystal structure of three EcMnmA/tRNA complexes corresponding to distinct catalytic states, it was first proposed in 2006 that EcMnmA proceeds *via* a persulfide-based mechanism involving two catalytic cysteines (Figure 1B), as in the case of *E. coli* U8-tRNA thiolase ThiI that possesses a rhodanese domain [31]: (1) a persulfide forms at a first catalytic cysteine of the enzyme, Cys199, via transpersulfuration reactions initiated by a cysteine desulfurase, (2) sulfur is liberated for nucleoside modification thanks to
a second catalytic cysteine, Cys102, (3) a reduction of the resulting disulfide closes the cycle [12] (see Figure 5 in [12]). Either a hydrosulfide-mediated mechanism was proposed (Figure 1B) or a direct sulfur transfer from the persulfide on Cys199 to the target nucleoside [12]. However, dithiothreitol (DTT) was used in the catalytic assays as a reductant, as an alternative to the physiological reductase that is required for the selective reduction of intermediates and for multiple turnovers. We have recently discussed in full detail the two main catalytic mechanisms of tRNA sulfurating enzymes and emphasized that the mechanism involving the direct attack of the persulfide formed on MnmA on the adenylated intermediate should be tested with IscS/cysteine as the sulfur source, in the absence of DTT, by monitoring the formation of one equivalent of products relative to the enzyme, because DTT reduces the persulfides formed on the desulfurase and transpersulfurases and generates hydrosulfide [32, 33]. Thus, in the presence of DTT, only the sulfide-mediated mechanism can be tested, with hydrosulfide being produced directly by persulfide reduction, without formation of a disulfide bond between the two catalytic cysteines. Unfortunately, no definitive conclusion about the catalytic mechanism can be drawn from the work of Numata et al. [12] because 1) enzyme assays using IscS/cysteine as the sulfur source in the absence of DTT have not been reported and 2) the possibility that hydrosulfide can reach the active site and serve directly as the nucleophile has not been checked using inorganic sulfide as a sulfur source, as was performed for *E. coli* ThiI [34].

A few years ago, two groups including ours have reported that EcMnmA is able to bind a [4Fe-4S] cluster [16, 29]. However, the cluster was found in our hands to be essential for the tRNA thiolation activity using a tRNA transcript as a substrate and inorganic sulfide as a sulfur source *in vitro* [16] (see the proposed catalytic mechanism in Figure 1C), while it was found to be inhibitory, also under *in vitro* conditions, using *E. coli* ΔiscS bulk tRNA as a substrate and cysteine together with cysteine desulfurase IscS as a sulfur source, in the presence of DTT [29]. To explore these contradictions, we report here new *in vitro* catalytic assays of EcMnmA before (apo-EcMnmA) and after cluster reconstitution (holo-EcMnmA) using, as a sulfur source, inorganic sulfide or IscS/cysteine, in the absence or presence of DTT.

Altogether, our results confirm that only holo-EcMnmA is able to catalyze thiolation of U34-tRNA in a tRNA transcript, using sulfide as a sulfur source. Moreover, cysteine/IscS could not serve as a sulfur source in the absence of a reductant and *E. coli* Δ*mnmA* bulk tRNA, containing an aminomethyl modification at C5, was not a substrate for apo/holo-EcMnmA, indicating that thiolation at C2 of U34 precedes modification at C5 in *E. coli* tRNAs.

# 2 Results

## 2.1 Preparation of holo-MnmA proteins

Since the procedure for reconstitution of the [4Fe-4S] cluster in EcMnmA described previously [16] was poorly reproducible, we tried to optimize the purification and reconstitution procedures of holo-
EcMnmA (see Material and Methods section). Shorter incubation with the reconstitution mixture (2-3 h instead of overnight) improved the amount of incorporated [4Fe-4S] cluster, as shown by the characteristic peak at 410 nm in the UV-visible spectrum (Figure 2A). However, some degradation of the [4Fe-4S] cluster occured during the subsequent purification on a gel filtration column, as shown by the absorption in the 550-650 nm region (Figure 2A). In the case of wild-type holo-EcMnmA, the Fe content of 2.05 ± 0.15 and the absorbance ratio at 410 nm and 280 nm of around 0.16 indicated incomplete occupation by the [4Fe-4S] cluster.

## 2.2 Inorganic sulfide can be used as a sulfur source *in vitro* by holo-EcMnmA, but not apo-EcMnmA

We first monitored the thiolation activity of apo and holo-EcMnmA on a tRNA transcript using inorganic sulfide as a sulfur source. After incubation of the enzyme in the presence of tRNA<sup>Glu</sup>, ATP, MgCl<sub>2</sub> and Na<sub>2</sub>S, the s<sup>2</sup>U content of the product was quantified after tRNA digestion using high performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS). Apo-EcMnmA could not thiolate a tRNA<sup>Glu</sup> transcript using inorganic sulfide as a sulfur source (Figure 3, sample 3), as reported previously [16]. In the same conditions, holo-MnmA was able to thiolate tRNA<sup>Glu</sup> (Figure 3, sample 6), with a number of cycles of 0.12. Therefore, our results confirm that the cluster is essential for the catalytic activity of EcMnmA on a tRNA transcript, using inorganic sulfide as a sulfur source.

## 2.3 Cysteine together with IscS cannot serve as a sulfur source for s<sup>2</sup>U thiolation by apo/holo-EcMnmA in the absence of DTT

In *E. coli*, the PLP-dependent cysteine desulfurase IscS, is responsible for the biosynthesis of all thionucleosides [35]. The question was raised whether EcMnmA could use IscS and cysteine as the sulfur source for tRNA thiolation *in vitro* because a dissociation constant of 5-10 µM was measured by surface plasmon resonance for the IscS/EcMnmA complex [29]. If this complex is able to catalyze sulfur transfer from cysteine towards the U34 target nucleoside, it is expected to be less efficient than the full sulfur relay system used by *E. coli* for biosynthesis of s<sup>2</sup>U34 that involves several transpersulfurases : TusA, the TusBCD complex and TusE [36].

We thus performed catalytic assays to know whether the IscS/EcMnmA/cysteine system, with EcMnmA in the apo- or the holo- state, could be active in U34-tRNA thiolation on a tRNA<sup>Glu</sup> transcript, in the presence or absence of DTT (Figure 3, samples 1, 2, 4 and 5). Our results show that apo-EcMnmA is not able to thiolate a tRNA<sup>Glu</sup> transcript using IscS/cysteine as a sulfur source, either in the absence or presence of DTT (Figure 3, samples 1 and 2). Moreover, the IscS/holo-MnmA complex showed 2-thiouridine synthesis activity using cysteine as a sulfur donor only in the presence of DTT (Figure 3, samples 4 and 5), i.e. through sulfide release in the medium, with a number of cycles of 0.16.
## 2.4 Bulk tRNA from an *E. coli* Δ*mnmA* strain is not a substrate of *EcMnmA*

Because tRNA transcripts were good substrates for holo-EcMnmA [16] and holo-TtMnmA [15], the C5-modification of U34 is not required for s²U34-tRNA synthesis. Interestingly, the MnmE•GidA complex could convert s²U into cmnm⁵s²U or nm⁵s²U, in a bulk tRNA from an *E. coli* Δ*mnmE* Δ*gidA* strain, in the presence of glycine or ammonium, respectively, indicating that the C5 modification can occur after the C2 thiolation of U34 [19]. Moreover, bulk tRNA was shown previously to be a substrate of several [4Fe-4S]-dependent tRNA thiolation enzymes using inorganic sulfide as a sulfur source. For example, *E. coli* holo-TtcA catalyzed s²C32 synthesis in an *E. coli* Δ*ttcA* bulk tRNA [37], *Methanoccus maripaludis* holo-TtuI catalyzed s⁴U8 synthesis in an *E. coli* Δ*thiI* bulk tRNA [38] whereas *Thermotoga maritima* and *Pyrococcus abyssi* TtuA catalyzed s²U54 synthesis in an *E. coli* Δ*ttcA* bulk tRNA lacking m⁵s²U [39]. Therefore, to gain further insights into the substrate specificity of the EcMnmA-catalyzed reaction *in vitro*, we tested whether an *E. coli* Δ*mnmA* bulk tRNA, which contains the (c)mnm modification at C5 [24, 27, 36], is a substrate of EcMnmA using IscS/cysteine as the sulfur source, in the absence or presence of DTT. However, no s²U, mnm⁵s²U or cmnm⁵s²U was formed, neither with apo- or holo-EcMnmA (Table 1), in the presence or absence of DTT, indicating that bulk tRNA from an *E. coli* Δ*mnmA* strain is not a substrate of EcMnmA.

## 2.5 The D99C variant of holo-EcMnmA is more active in tRNA thiolation than wild-type EcMnmA *in vitro*

We mutated the conserved aspartate of the catalytic motif, Asp99, into cysteine in order to enhance binding of the [4Fe-4S] cluster. The iron content of the D99C-EcMnmA variant of 3.1 ± 0.4, together with the UV-visible spectrum and the absorbance ratio at 410 nm and 280 nm of 0.35 (Figure 2B) indicated the presence of a well-reconstituted [4Fe-4S] cluster. Activity tests were performed using ATP, MgCl₂, inorganic sulfide as a sulfur source and a tRNA*Glu* transcript as a substrate (Figure 4). Like apo-EcMnmA, apo-D99C-EcMnmA could not catalyze s²U formation in these conditions (Figure 4, samples 1 and 2). The holo-D99C-EcMnmA variant showed enhanced 2-thiouridylation activity compared to the wild-type enzyme, with a number of cycles of 0.32 (Figure 4 samples 5 and 6). In addition, both the wild-type and the D99C variant of holo-EcMnmA showed little catalytic activity in the absence of a sulfur donor (Figure 4, samples 3 and 4).

## 2.6 Anaerobically purified EcMnmA contains a [4Fe-4S] cluster

To check if EcMnmA is able to bind a [4Fe-4S] cluster *in vivo*, we overexpressed EcMnmA in the presence of the pBD1282 plasmid that overexpresses the *Azotobacter vinelandii* ISC machinery to facilitate the incorporation of [Fe-S] cluster in proteins [40]. After cells disruption with lysozyme, the protein was purified anaerobically on a Nickel affinity column without chemical cluster reconstitution.
The UV-visible spectrum of such anaerobically purified EcMnmA shows a partially degraded [4Fe-4S] cluster (Figure 2C), with 0.1 Fe atom incorporated per monomer.

## 2.7 The D99C-EcMnmA variant does not catalyze s²U synthesis in tRNA *in vivo*

We then wondered whether expression of the D99C-EcMnmA variant could complement the *E. coli* Δ*mnmA* strain. To this end, bulk tRNA was extracted from the *E. coli* Δ*mnmA* strain and from this strain complemented by the *mnmA* and D99C-*mnmA* genes, and the modified nucleosides were quantified by HPLC-MS/MS (Table 2). The results show that no cmnm⁵s²U or mnm⁵s²U was formed *in vivo* in the *E. coli* Δ*mnmA* strain complemented by the D99C-*mnmA* gene.

# 3 Discussion

Inorganic sulfide can be directly used as a sulfur source to test the *in vitro* thiolation activity of [4Fe-4S]-dependent tRNA thiolases [15, 33, 38, 39, 41] (Figure 1C). Sulfur in the form of persulfide can also be generated *in vitro* using cysteine and cysteine desulfurase [14, 27, 29]. However, in that case, if DTT is employed as a cosubstrate to allow multiple cycles, it reductively cleaves the persulfide intermediates [32, 42] and releases the S atom in the form of hydrosulfide [33]. Therefore, inorganic sulfide can also be used as a sulfur source to know whether apo-MnmA can use a hydrosulfide-based mechanism for catalyzing tRNA thiolation (Figure 1B).

Our activity tests using, as a sulfur source, inorganic sulfide or IscS/cysteine in the presence of DTT, confirm our previous results [16] showing that holo-EcMnmA, but not apo-MnmA, can thiolate a tRNA<sup>Glu</sup> transcript, in contradiction with the report stating that the cluster is an inhibitor of the thiolation activity of EcMnmA [29]. The low number of cycles of 0.12 for holo-EcMnmA with sodium sulfide can be explained by the low stability of the cluster of EcMnmA, which leads to a fast inactivation of the enzyme. When L-cysteine was used as a sulfur source in the presence of all transpersulfurases of the s²U34-tRNA biosynthetic pathway, the number of cycles was very similar (0.1) [36]. In that case, the activity was most likely due to the release of hydrosulfide in the medium because DTT was used in these experiments, and it can reduce persulfides formed on the cysteine desulfurase and transpersulfurases. However, this reported activity is in contradiction with the fact that we did not detect any catalytic activity for apo-EcMnmA, with sodium sulfide as a sulfur source, which we take as a proof to exclude a persulfide-based mechanism involving hydrosulfide as a nucleophile as described in Figure 1B. Likewise, to avoid reduction of the persulfide on IscS, the persulfuration reaction between IscS and apo-MnmA needs to be tested in the absence of a reductant. In this case, the formation of no more than one equivalent of product per MnmA enzyme is expected since a reductant is needed for multiple turnovers [33]. Since no activity was observed for apo-EcMnmA with IscS/cysteine in the absence of DTT, it is impossible to conclude about the potential persulfide transfer between IscS and apo-MnmA. Using
IscS/cysteine as a sulfur source, in the presence of DTT, the number of cycles of holo-MnmA was 0.16, similar to that obtained with Na₂S as a sulfur source.

Remarkably, holo-D99C-EcMnmA showed greater efficiency than wild-type holo-EcMnmA in catalyzing s²U-tRNA formation *in vitro*. This enhanced activity is likely due to the [4Fe-4S] cluster being coordinated by three cysteines, which results in a more stable association with the protein compared to coordination by two cysteines and one aspartate. This observation supports the critical role of the [4Fe-4S] cluster in the thiolation process. In agreement with the reconstitution of a [4Fe-4S] cluster within aerobically purified EcMnmA, anaerobically purified EcMnmA was shown to bind an iron-sulfur cluster mainly as the [4Fe-4S] form (Figure 2C). Interestingly, some activity of holo-EcMnmA and holo-D99C-EcMnmA in the absence of a sulfur source was observed, likely due to the presence of a small amount of [4Fe-5S] cluster formed during the chemical reconstitution of the cluster, which can release sulfur during the activity test, as previously reported for MmNcsA [17].

Previous isolation of *E. coli* mutants containing only the mnm⁵ or s² modification at U34 [43, 44] indicated that these modifications can occur independently of each other. It was thus concluded that thiolation may precede or follow the synthesis of the side chain at position 5 [24]. However, we show here that bulk tRNA from the *E. coli* Δ*mnmA* strain is not a substrate of apo/holo-EcMnmA (Table 1). The absence of s²U synthesis suggests that there is very little unmodified tRNA substrate (without C5 modifications of U34), in the *E. coli* Δ*mnmA* strain. Moreover, the absence of mnm⁵s²U and cmnm⁵s²U indicates that the modification at C5 of U34, which is present in bulk Δ*mnmA* tRNA [24, 27, 36], prevents thiolation at C2. This suggests that thiolation of U34 at C2 precedes modification at C5 in *E. coli* tRNAs. Similarly, bulk tRNA from the *E. coli* Δ*iscS* strain that also contains a C5 modification at U34 [45] is most likely not a substrate of *E. coli* MnmA. This could explain why the s²U activity was very low both with apo and holo-EcMnmA when bulk tRNA from the *E. coli* Δ*iscS* strain was used as a potential substrate, with IscS/cysteine being used as a sulfur source in the presence of DTT [29]. Interestingly, the modification order, at C2 then at C5, was previously reported for apo-BsMnmA [14] despite a very low catalytic efficiency of the enzyme. The substrate preference of EcMnmA and BsMnmA for tRNAs containing no cmnm⁵/mnm⁵ modification at U34 can be rationalized by the active site structure of EcMnmA, which cannot accommodate the cmnm⁵ modification at U34 (Figure5A), as noticed previously [14]. Indeed, the superposition of *E. coli* tRNA<sup>Gln</sup> containing cmnm⁵U34 [46] with tRNA<sup>Glu</sup> from the crystal structure of the EcMnmA/tRNA<sup>Glu</sup> complex [12] shows that the bulky cmnm⁵ modification at U34 clashes with conserved Gln151 (Figure 5A). Therefore, we conclude from our study that, in wild-type *E. coli* strain, in which both modifications at C2 and C5 take place, there is an order of the modifications occuring at U34, with thiolation at C2 preceding modification at C5 (Figures 5B and 5C).

Purification of EcMnmA under anaerobic conditions showed that EcMnmA binds a [Fe-S] cluster *in vivo*. Unexpectedly, the D99C-EcMnmA variant failed to complement the Δ*mnmA* strain, as it did not
restore mnm⁵s²U or cmnm⁵s²U biosynthesis (Table 2). This result supports an essential role for the aspartate that coordinates the [4Fe-4S] cluster for *in vivo* U34-tRNA thiolation. We currently have no explanation for the apparent requirement for a "softer" ligand (aspartate rather than cysteine) *in vivo*, a requirement that is not recapitulated by our *in vitro* experiments.

Further investigations of EcMnmA should include *in vitro* assays performed without DTT and using the full set of proteins involved in sulfur transfer from cysteine to mimic the *in vivo* system [36], with quantification of product relative to enzyme molecule [33]. Complementary *in vivo* studies are also needed to determine the functional consequences of substituting a cysteine ligand with aspartate for [4Fe-4S] cluster coordination. A possibility is that MnmA could be involved in sensing the sulfur environment in the cell via its cluster, which would need a soft coordination. Future investigations should address how cluster stability affects EcMnmA activity and clarify the role of the Tus relay proteins in guiding sulfur transfer from IscS to EcMnmA.

# 4 Material and methods

The obtention of the *E. coli* Δ*mnmA* strain was reported in [16]. The *E. coli* tRNA<sup>Glu</sup> transcript was bought from Horizon Discovery Biosciences. The pET28a plasmids encoding EcMnmA (pML23) and D99C-MnmA (pML62) were described previously [16, 47]. These constructions enable expression of EcMnmA and the variant with an N-terminal histidine tag, cleavable by human rhinovirus 3C (H3C) and tobacco etch virus (TEV) proteases, respectively.

## 4.1 Preparation of bulk tRNA

The pET28 plasmids encoding EcMnmA and D99C-EcMnmA were transformed into *E. coli* BL21(DE3) Δ*mnmA* competent cells. Bulk tRNA was purified from the *E. coli* Δ*mnmA* strain and the complemented strains as follows. One colony was used to inoculate 100 mL of LB medium supplemented with kanamycin (50 mg/mL) or without antibiotics for the Δ*mnmA* strain used as control. 15 mL of this culture grown overnight at 37 °C was used to inoculate 2 L of LB medium supplemented with kanamycin. After overnight incubation at 37 °C, the cells were centrifuged and the pellets resuspended in 10 mL of 1 mM Tris pH 7.4, 10 mM Mg-acetate. After addition of 15 mL water-saturated phenol to each pellet and incubation overnight at room temperature on a wheel, the mixture was centrifuged at 5,500 rpm for 20 min at 25 °C. The supernatants were collected, incubated with 0.1 volume of 5 M NaCl and 2 volumes of 100 % ethanol (HPLC grade). After centrifugation at 5,500 rpm for 20 min at 4 °C, the pellets were resuspended with 5 mL of 1 M NaCl and centrifuged at 5,500 rpm for 20 min at 4 °C. The supernatants were mixed with 2.5 volumes of 100 % ethanol and incubated at - 20 °C for 4 h. After centrifugation at 5,500 rpm for 20 min at 4 °C, the pellets were resuspended in 5 mL water, then loaded at 2.5 mL/min onto a NucleoBond Xtra column (Macherey Nagel) pre-equilibrated with buffer A (100 mM Tris-H<sub>3</sub>PO<sub>4</sub>, pH 6.3, 0.4 M KCl) on an ÅKTA system. After elution with 40 % buffer B (100 mM Tris-H<sub>3</sub>PO<sub>4</sub>, pH
6.3, 2 M KCl), the fractions containing tRNAs were pooled and incubated with one volume of isopropanol overnight at – 20 °C. After centrifugation at 5,500 rpm for 20 min at 4 °C, the pellets were resuspended with 10 mL of cold 70 % ethanol solution. After centrifugation at 5,500 rpm for 20 min at 4°C, the pellets were dried and resuspended in 400 µL water. The concentration of bulk tRNA was determined using a Nanodrop apparatus.

## 4.2 Overexpression, purification of EcMnmA and reconstitution of the [4Fe-4S] cluster

The overexpression of wild-type EcMnmA construct was previously described [16]. Cell pellets were resuspended in 25 mM Tris-HCl pH 7.5, 500 mM NaCl, 10% glycerol, containing RNase A (2 µg mL⁻¹), benzonase (1.6 U mL⁻¹), lysozyme (0.3 mg mL⁻¹), 1 mM PMSF (Phenyl Methyl Sulfonyl Fluoride), 5 mM β-mercaptoethanol and lysed by sonication. The lysate was centrifuged at 40,000 rpm at 8 C for 30 min to remove cell debris and the supernatant loaded on an immobilized metal affinity Ni-NTA column (HisTrap 5 ml, GE Healthcare) that was equilibrated in 25 mM Tris-HCl (pH 7.5), 300 mM NaCl, 10 % glycerol and eluted with a linear gradient of 0–500 mM imidazole. The protein fractions were collected, dialyzed against 1 L of 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM β-mercaptoethanol, in the presence of the H3C protease. The protein was treated with 100 mM dithionite and 100 mM EDTA to remove the residual cluster and further purified using a gel filtration column (Hiload 26/60 Superdex 200, GE Healthcare) equilibrated with 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 % glycerol. The purified proteins were concentrated with an Amicon Ultra filter device (10 kDa cutoff), frozen in liquid nitrogen and stored at –80°C. The reconstitution of the [4Fe-4S] cluster and subsequent purification of holo-MnmA were performed in a glove box containing <0.5 ppm O₂. 100 µM as-purified MnmA (wild-type or D99C variant) was treated with 10 mM dithiothreitol for 10 min. Then a 6-fold molar excess of ferrous ammonium sulfate and L-cysteine, as well as 2 µM *E. coli* cysteine desulfurase CsdA, were added, and incubation was pursued at room temperature for 2-3 h. After centrifugation for 20 min at 10,000 rpm, the holo-proteins were loaded onto an analytical gel filtration column (10/300 GL Superdex 200 Increase) equilibrated in 50 mM Tris-HCl pH 7.5, 150 mM NaCl. The colored fractions containing the holo-proteins were then concentrated using an Amicon Ultra filter device (10 kDa cutoff). After spectral analysis, the protein was frozen in liquid nitrogen and kept at – 80 °C. The same purification protocol was used for D99C-MnmA except that the H3C protease was replaced by the TEV protease to cleave the histidine tag.

For anaerobic purification of EcMnmA with proper [4Fe-4S] incorporation, the pET28A-EcMnmA plasmid was co-transformed into competent BL21(DE3) *E. coli* cells with the *iscS-iscU-iscA-hscB-hscA*-fdx genes containing the [4Fe-4S] biosynthetic machinery from *A. vinelandii* within the ampicillin-resistant, ara-inducible pBD1282 plasmid [48]. Cell growth was performed as described above except that ampicillin (50 µg/mL) was added to the cultures. Cells were broken within the glovebox using lysozyme (0.3 mg/mL).

The Fe content in the holo-proteins was quantified using the Fish procedure [49].
## 4.3 Catalytic activity

The activity tests of apo- and holo-proteins were performed in a glove box (MBraun) containing less than 0.5 ppm O₂. After defrosting, the apo-proteins were degassed, then incubated with 10 mM DTT for 30 min. After centrifugation for 10 min at 10,000 rpm, the apo-proteins were loaded onto an analytical gel filtration column (10/300 GL Superdex 200 Increase), equilibrated in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, to get rid of the reductant. The fractions containing the apo-proteins were then concentrated with an Amicon Ultra filter device (10 kDa cutoff), pooled and used immediately for the activity tests. Apo/holo-EcMnmA/D99C-EcMnmA (5 µM) was incubated with 15 µM tRNAGlu or *E. coli* bulk tRNA (100 µg) in the presence of ATP (1 mM), MgCl₂ (2.5 mM), and with IscS (2 µM) together with L-cysteine (0.1 mM) or Na₂S (1 mM) as a sulfur source, in the presence or absence of DTT (3 mM). Quantification of uridine modifications in the tRNA product was performed using HPLC coupled MS/MS, as previously described [16], using s²U, mnm⁵s²U and cmnm⁵s²U standards.

**Acknowledgments.** This work was supported by the Centre National de la Recherche Scientifique, Sorbonne Université as well as the "Initiative d'Excellence" program from the French State (Grant "DYNAMO", ANR-11-LABX-0011-01) and the French National Research Agency (grant "sulfo-tRNA" ANR-22-CE44-0012). This project has received funding from the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No 101034407.
Tables

<table><caption>**Table 1: In vitro Activity of EcMnmA using E. coli ΔmnmA bulk tRNA as a substrate.**</caption><thead><tr><th>Sample</th><th>s<sup>2</sup>U (pmoles)</th><th>cmnm<sup>5</sup>s<sup>2</sup>U (pmoles)</th><th>mnm<sup>5</sup>s<sup>2</sup>U (pmoles)</th></tr></thead><tbody><tr><td>apo-EcMnmA no DTT</td><td>0.45 ± 0.14</td><td>0.00</td><td>0.005 ± 0.01</td></tr><tr><td>apo-EcMnmA + DTT</td><td>0.03 ± 0.11</td><td>0.00</td><td>0.005 ± 0.01</td></tr><tr><td>holo-EcMnmA no DTT</td><td>0.78 ± 0.10</td><td>0.00</td><td>0.01 ± 0.01</td></tr><tr><td>holo-EcMnmA + DTT</td><td>0.09 ± 0.16</td><td>0.00</td><td>0.00 ± 0.01</td></tr></tbody></table>

The s²U, cmnm⁵s²U and mnm⁵s²U contents in *E. coli* ΔmnmA bulk tRNA (100 µg) were quantified by HPLC-MS/MS after incubation with apo/holo-EcMnmA using IscS/cysteine as a sulfur source in the presence or absence of DTT. The results are the mean of three measurements. The positive control indicating that holo-EcMnmA is catalytically active in this experiment is shown in Figure 3, sample 5.

<table><caption>**Table 2: In vivo Activity of EcMnmA and D99C-MnmA.**</caption><thead><tr><th>Strain</th><th>cmnm<sup>5</sup>s<sup>2</sup>U (pmoles)</th><th>mnm<sup>5</sup>s<sup>2</sup>U (pmoles)</th></tr></thead><tbody><tr><td>ΔmnmA</td><td>0.015 ± 0.03</td><td>0.05 ± 0.05</td></tr><tr><td>ΔmnmA + mnmA</td><td>39.6 ± 1.04</td><td>510.4 ± 58.3</td></tr><tr><td>ΔmnmA + D99C-mnmA</td><td>0.14 ± 0.25</td><td>0.03 ± 0.01</td></tr></tbody></table>

The mnm⁵s²U and cmnm⁵s²U contents in *E. coli* ΔmnmA bulk tRNA were quantified by HPLC-MS/MS after complementation with the *mnmA* and D99C-*mnmA* genes. The results are the mean of three experiments.
<figure><img src="image_2.png" /><figcaption>**Figure 1:** Two possible mechanisms for U34-tRNA sulfuration by EcMnmA. **A** Activation of the C2 position of the target base U34 by ATP to form an adenylated intermediate is common to both mechanisms. **B** Persulfide-based mechanism involving two catalytic cysteines and a hydrosulfide nucleophile, as proposed by Numata et al. [12]. Ad stands for ‘adenylate’. **C** [4Fe-4S]-dependent mechanism involving the formation of a [4Fe-5S] catalytic intermediate.</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>**Figure 2.** UV-visible spectra of wild-type and D99C-holoMnmA. **A** UV-visible spectra of wild-type EcMnmA (20 µM in 50 mM Tris pH 7.5; 150 mM NaCl) before (apo-EcMnmA, t=0), after cluster reconstitution (holo-EcMnmA, t=90 min) and after cluster reconstitution followed by gel filtration (holo-EcMnmA after gel filtration). **B** UV-visible spectrum of D99C-holo-MnmA after gel filtration (20 µM in 25 mM Tris-HCl pH 7.5, 150 mM NaCl). **C** UV-visible spectrum of wild-type EcMnmA (750 µM) after expression in the presence of the *isc* plasmid and anaerobic purification.</figcaption></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>
<figure><img src="image_7.png" /><figcaption>**Figure 3:** IscS/Cysteine as a potential sulfur source for the EcMnmA-catalyzed thiolation. Apo/holo-EcMnmA was incubated with tRNA<sup>Glu</sup> in the presence of ATP, MgCl<sub>2</sub> and with IscS/cysteine or Na<sub>2</sub>S as a sulfur source, in the presence or absence of DTT.</figcaption></figure>

<table><tr><td>Na<sub>2</sub>S</td><td></td><td></td><td>+</td><td></td><td></td><td>+</td></tr><tr><td>IscS + Cys</td><td>+</td><td>+</td><td></td><td>+</td><td>+</td><td></td></tr><tr><td>DTT</td><td></td><td>+</td><td></td><td></td><td>+</td><td></td></tr><tr><td>EcMnmA</td><td>apo</td><td>apo</td><td>apo</td><td>holo</td><td>holo</td><td>holo</td></tr></table>
<figure><img src="image_8.png" /><figcaption>**Figure 4:** Holo-D99C-EcMnmA is more active than wild-type holo-EcMnmA using Na₂S as a sulfur source and a tRNA<sup>Glu</sup> transcript *in vitro*. Apo/holo-EcMnmA or apo/holo-D99C-EcMnmA was incubated with tRNA<sup>Glu</sup> in the presence of ATP, MgCl₂ and Na₂S as a sulfur source and in the absence of DTT.</figcaption></figure>

<table><tr><td>Na₂S</td><td>+</td><td>+</td><td>-</td><td>-</td><td>+</td><td>+</td></tr><tr><td>EcMmA</td><td>apo</td><td></td><td>holo</td><td></td><td>holo</td><td></td></tr><tr><td>D99C-EcMnmA</td><td>-</td><td>apo</td><td></td><td>holo</td><td></td><td>holo</td></tr></table>
<figure><img src="image_9.png" /><figcaption>**Figure 5: Thiolation at C2 of U34-tRNA precedes modification at C5 in E. coli.** A The modified nucleoside cmnm⁵U34 cannot be accommodated in the active site of EcMnmA. The anticodon loop of tRNAGln with the cmnm⁵s²U34 modified nucleoside (PDB code 4JYZ) was superposed to that of tRNAGlu from the crystal structure of EcMnmA in complex with tRNAGlu in the pre-reaction state (PDB code 2DET) with an rmsd of 0.336 Å for 12,243 atoms. EcMnmA is in orange, U34 of tRNAGlu in cyan and cmnm⁵s²U34 of tRNAGln in magenta. The superposition shows steric hindrance between the carboxylate of the cmnm⁵ group of U34 and conserved Gln151. B Order of modifications at C2 and C5 positions of U34 in E. coli tRNAs. C Biosynthetic pathway of mmmn⁵s²U34 in tRNALys, tRNAGlu and tRNAGln in E. coli. First, thiolation at carbon 2 of U34 is catalyzed by MnmA using ATP to activate the target position. Second, the MnmE/MnmG complex modifies the C5 position of U34 using four different cofactors, with flavine adenine dinucleotide (FAD) and nicotinamide adenine dincleotide (NADH) binding to MnmG and methylenetetrahydrofolate (MTHF) and GTP to MnmE. The MnmE/MnmG complex catalyzes the addition of the carboxymethylaminomethyl (cmnm) and aminomethyl (nm) groups to position 5 of U34 using glycine and ammonium, respectively, depending on the growth conditions. Finally, the FAD-dependent activity of the bifunctional enzyme MnmC transforms cmnm⁵s²U into nm⁵s²U, whereas the S-adenosyl-L-methionine (SAM)-dependent methyltransferase activity of MnmC transforms nm⁵s²U into mnm⁵s²U using SAM as the methyl donor.</figcaption></figure>
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