# Structural and biochemical insights into the broad-spectrum TET enzyme from Methanocaldococcus jannaschii reveal the basis of substrate specificity in M42 aminopeptidases

Joaquin Atalah, Hind Basbous, Gregory Effantin, Sylvie Kieffer-Jaquinod, Guy Schoehn, Eric Girard, Bruno Franzetti

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Joaquin Atalah, Hind Basbous, Gregory Effantin, Sylvie Kieffer-Jaquinod, Guy Schoehn, et al.. Structural and biochemical insights into the broad-spectrum TET enzyme from Methanocaldococcus jannaschii reveal the basis of substrate specificity in M42 aminopeptidases. Journal of Molecular Biology, 2026, 438 (3), pp.169596. 10.1016/j.jmb.2025.169596. hal-05537817

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# Structural and Biochemical Insights into the Broad-Spectrum TET Enzyme From *Methanocaldococcus jannaschii* Reveal the Basis of Substrate Specificity in M42 Aminopeptidases

Joaquin Atalah<sup>a</sup>, Hind Basbous<sup>a</sup>, Gregory Effantin<sup>a</sup>, Sylvie Kieffer-Jaquinod<sup>b</sup>, Guy Schoehn<sup>a</sup>, Eric Girard<sup>a</sup>, and Bruno Franzetti<sup>a,\*</sup>

<sup>a</sup> - Univ Grenoble Alpes, CNRS, CEA, IBS, Grenoble, France

<sup>b</sup> - INSERM, CEA, Univ Grenoble Alpes, UMR BioSanté U1292, CNRS, FR2048 Grenoble, France

Correspondence to Bruno Franzetti: bruno.franzetti@ibs.fr (B. Franzetti)

https://doi.org/10.1016/j.jmb.2025.169596

Edited by Zhi-Jie Liu

## Abstract

TET peptidases of the M42 family are ~500 kDa hollow dodecameric complexes ubiquitous in prokaryotes. These enzymes act as strict aminopeptidases, catalyzing the removal of N-terminal amino acids from peptides. A common feature of M42 TET aminopeptidases characterized to date is their marked substrate preference for a limited subset of amino acids. Unlike other hyperthermophilic archaea studied so far, the autotrophic archaeon *Methanocaldococcus jannaschii* possesses only a single gene encoding an M42 peptidase. This enzyme, named MjTET, is the first reported M42 peptidase to exhibit broad amino acid specificity, including activity on aromatic residues. To assess their peptide degradation efficiencies, the catalytic constants of MjTET were compared to those of its close analogs from *Pyrococcus horikoshii*. The specialized TETs from *P. horikoshii* displayed higher catalytic efficiencies than the generalist MjTET, likely reflecting the reliance of Thermococcales on peptide fermentation for energy. Additionally, the structure of MjTET was resolved to 3 Å using cryo-EM and compared with the available models of the four *P. horikoshii* TETs to identify features underlying substrate specificity. This analysis, combined with mutagenesis studies, revealed a previously uncharacterized loop in the catalytic domain that contributes to substrate discrimination. Collectively, these findings show that substrate specificity in TET enzymes arises from a complex interplay of tertiary structure, oligomeric assembly, and electrostatic surface potential.
**Importance:** This study first reported a novel TET peptidase from Methanogenic hyperthermophilic archaea. Its enzymatic properties compared to the specialized TET enzyme characterized so far from heterotrophic archaea suggest a link with autotrophy. It also represents an important step in explaining the structural features guiding substrate specificity.

© 2025 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

## Introduction

Aminopeptidases are a broad class of enzymes that hydrolyze peptide bonds between amino acids in peptide chains. They participate in a wide range of cellular processes, including protein degradation and post-translational modification of proteins and peptides [1,2]. Many aminopeptidases are metalloenzymes, containing catalytic metal ions

coordinated by active-site residues, and can be divided into different families within the MH clan, according to the classification proposed by the MEROPS database which reflects their evolutionary relationships [3].

Over the past few decades, several aminopeptidases from the M42 and M18 families have been found to self-assemble into dodecameric complexes of approximately half a
megadalton [4]. They were named TET enzymes due to the tetrahedral shape of the assembled particles. The first TET complex to be characterized was purified from a halophilic archaeon during a search for protein quality-control machines involved in low-salt stress [5]. This newly identified class of cage-forming proteases attracted interest because of its widespread presence across diverse organisms, with homologs found in all three domains of life.

Structural studies have shown that TET dodecamers share a conserved architecture: Six anti-parallel dimers assemble into a tetrahedral complex featuring four large pores formed at the interface of six subunits on each face. Inside, four catalytic chambers are positioned at the tetrahedron's vertices, each containing three active-sites formed by the convergence of three monomers [4]. This architecture is strikingly different from the barrel-shaped one adopted by other cytosolic compartmentalized peptidases.

Although different types of TET enzymes have been found in archaea, bacteria and eukarya, all M42 TETs characterized to date are divalent metal cation-activated aminopeptidases capable of degrading oligopeptides into single amino acids [6–12]. However, enzymatic studies revealed that they displayed marked substrate preferences. It has also been found that different versions of these enzymatic complexes can coexist in the cytosol of the same organism. This aspect of TET biochemistry has been mostly studied in *Pyrococcus horikoshii*, a heterotrophic, deep-sea, hyperthermophilic euryarchaeon that contains four distinct TET complexes: PhTET1, a glutamyl-aspartyl aminopeptidase; PhTET2, a leucyl aminopeptidase with activity against neutral amino acids; PhTET3, a lysyl aminopeptidase; and PhTET4, a strict glycyl aminopeptidase [6–9,13]. Similar patterns of multiple TET enzymes have been observed in other Thermococcales species.

*Methanocaldococcus jannaschii* is another archaeon inhabiting abyssal hydrothermal environments. It is an autotroph capable of carbon fixation through methanogenesis and contains a single gene encoding a putative M42 peptidase in its genome. This observation raised questions about the identity and substrate specificity of this enzyme and how it differs from the TET peptidases found in hyperthermophilic archaea with multiple copies of TET, such as *P. horikoshii*.

In this study, we purified and characterized the recombinant M42 peptidase from *M. jannaschii* (MjTET), identifying it as a broad-spectrum, promiscuous aminopeptidase. To explore how the presence of a single “generalist” TET in *M. jannaschii* contrasts with the four “specialized” TETs in *P. horikoshii*, we compared the kinetic performance of MjTET with those of PhTET2 and PhTET3, which share partially overlapping substrate specificities. Our findings suggest that

the contrasting strategies—employing a single, generalist TET, versus multiple specialized variants—reflect the differences in lifestyles and carbon metabolism of these two archaea. Additionally, we determined the cryo-EM structure of the recombinant MjTET peptidase. Through a detailed comparative analysis of its atomic model and the structures of the four PhTETs, in addition to the study of MjTET mutants, we identified structural features that are consistent with their respective substrate selectivity and proposed molecular mechanisms underlying the observed differences in their biochemical properties. These findings shed light on how structural and biochemical differences in TET enzymes correlate with the metabolic adaptations of thermophilic archaea, offering new insights into enzyme evolution and functional specialization.

## Results

### MJ0555 gene encodes a tetrahedral dodecameric protein

In order to study the structure and function of an uncharacterized TET enzyme, we cloned and recombinantly expressed the *MJ0555* gene, identified as the single putative TET peptidase present in the genome of *M. jannaschii*. The corresponding heterologous protein was purified to homogeneity. When analyzed by gel filtration chromatography, the MJ0555 protein displayed an apparent molecular mass of about 460 kDa (Figure 1A). Negative stain electron microscopy images of purified MJ0555 samples showed typical tetrahedral particles with dimensions similar to those previously determined for the different M42 TET peptidases (Figure 1B). MJ0555 was therefore assigned to the TET family and named MjTET. Sequence alignment with the well-characterized TETs from *P. horikoshii* revealed conservation of the active-site residues (Supplementary Figure S1).

### MjTET is an aminopeptidase with broad substrate specificity

The amino acid selectivity of MjTET was investigated using chromogenic and fluorogenic monoacyl substrates. As shown in Figure 2, the enzyme acted on a broad range of aminopeptidase substrates. Interestingly, although slightly higher amidolytic activity was observed against glutamine, when the standard deviation of the measurements was taken into account, MjTET showed equivalent activity toward glutamine, methionine, and leucine. This indicates that MjTET is a true broad-range TET aminopeptidase, with no clear preference for a single amino acid type. Significant activity was also measured against lysine, serine, alanine, and arginine, and non-negligible activity was found against tyrosine,
<figure><img src="image_3.png" /><figcaption>**Figure 1.** MjTET is a dodecameric tetrahedral enzyme. (A) Size-exclusion chromatography (Superose-6 column) from the final purification step of MjTET, showing a major peak corresponding to an estimated molecular weight of ~460 kDa, consistent with the expected size for this enzyme class and in agreement with a dodecamer of the predicted MJ0555 gene product. (B) Negative-stain EM image of purified MjTET at 0.1 mg/mL. The particles show the characteristic tetrahedral shape of TET aminopeptidases. Scale bar: 20 nm.</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>**Figure 2.** MjTET substrate specificity. Different monoacyl-pNA and –AMC substrates were incubated in the presence of MjTET at 80 °C and pH 8.0, and the activity was calculated in order to estimate the relative specificity toward each amino acid.</figcaption></figure>

phenylalanine, isoleucine and valine. Significantly lower activities were detected with threonine, tryptophane, glutamate, histidine, glycine and asparagine. Proline, aspartate and cysteine were the only residues for which no activity was detected. Additionally, no activity was detected toward N-terminally blocked residues or D-configured residues, as demonstrated by Ac-Leu-pNA and D-Leu-pNA substrates, respectively.

## Influence of pH, metal ions, and temperature on leucine aminopeptidase activity of MjTET

Since MjTET showed high activity toward Leu-pNA, this substrate was used to determine the optimal conditions for MjTET aminopeptidase activity. The effect of pH on MjTET aminopeptidase activity was investigated (Figure 3A), revealing that MjTET exhibits its
<figure><img src="image_5.png" /><figcaption>**Figure 3.** Influence of pH, metal ions, and temperature on MjTET aminopeptidase activity. (A) pH dependence of leucine aminopeptidase (LAP) activity at 75 °C and 0.1 mM CoCl₂. Diamond mark: reaction performed in PIPES buffer. Triangles: reactions in HEPES buffer; Squares: reactions in CHES buffer. (B) Temperature dependence of LAP activity. Error bars indicate standard deviations of three replicates. (C) LAP activity at 80 °C in the absence of metals (WM, without metals) or in the presence of different metal ions. (D) Effect of Co²⁺ concentration on LAP activity at 80 °C in 50 mM HEPES buffer (pH 8.0). (E) Influence of 0.1 mM Co²⁺ on LAP activity at two different temperatures, showing that cobalt is required for activity across the tested range.</figcaption></figure>

higher activity at pH 8.0, while maintaining approximately 80% of its activity at pH 7 and around 75% at pH 9. Activity rapidly declined at more alkaline pH values, with almost no activity detected at pH 10. The temperature dependence of MjTET activity was also assessed. As shown in Figure 3B, aminopeptidase activity peaked at 85 °C. No activity was detected under mesophilic conditions below 50 °C.

Since divalent metal ions are known to modulate the activity of aminopeptidases, the effect of several metals on MjTET activity was tested. As shown in Figure 3C, aminopeptidase activity increased only in the presence of cobalt ions. In contrast, a significant inhibition was observed when 0.1 mM ZnCl₂ was added to the reaction mixture. Other metal cations tested also had a slightly inhibiting effect. Therefore, MjTET can be characterized as a cobalt-activated enzyme.

The effect of the concentration of cobalt used was therefore studied. When the concentration was increased from 0.01 mM to 1 mM, maximum activity was reached at 0.1 mM, with activity gradually decreasing as Cobalt concentration was further increased (Figure 3D). The presence of cobalt in the reaction significantly enhanced

measured activity compared to controls with no added cobalt, both at 75 °C and 50 °C (Figure 3E).

## Kinetic parameters

To evaluate the efficiency of a “specialized,” narrow-substrate-range TET compared to a “generalist,” wide-substrate-range TET, we assessed the performance of MjTET in contrast to its analogs PhTET2 and PhTET3 by calculating the kinetic parameters for their respective preferred substrates under identical reaction conditions. PhTET1 and PhTET4 were excluded from this comparison as their respective monoacyl substrates are not hydrolyzed by MjTET. The results (Table 1) reveal that both specialized TETs exhibit higher *k*<sub>cat</sub> values than MjTET for their preferred substrates. Notably, MjTET demonstrated a lower *K*<sub>M</sub> for Leu-pNA compared to PhTET2, indicating stronger substrate affinity; however, the catalytic efficiency (*k*<sub>cat</sub>/*K*<sub>M</sub>) for PhTET2 was still more than twofold higher. Similarly, for Lys-pNA hydrolysis, the catalytic efficiency of PhTET3 was an order of magnitude greater than that of MjTET. Collectively, these results suggest that substrate specialization is accompanied by enhanced catalytic efficiency.
<table><caption>Table 1 Kinetic performance of the three enzymes was compared in equal conditions of pH 8 and 75 °C. The kinetic parameters were calculated with a non-linear least-squares fitting of the experimental data of reaction rates as a function of substrate concentration.</caption><thead><tr><th>Enzyme</th><th>Substrate</th><th>Apparent K<sub>M</sub> (mM)</th><th>k<sub>cat</sub> (s<sup>-1</sup>)</th><th>k<sub>cat</sub>/K<sub>M</sub></th></tr></thead><tbody><tr><td>MjTET</td><td>Leu-pNA</td><td>0.2426 (±0.0237)</td><td>7213 (±872)</td><td>29,734 (±4618)</td></tr><tr><td>PhTET2</td><td>Leu-pNA</td><td>0.4360 (±0.0441)</td><td>31,422 (±1323)</td><td>72,067 (±7894)</td></tr><tr><td>MjTET</td><td>Lys-pNA</td><td>1.3149 (±0.1289)</td><td>7120 (±303)</td><td>5415 (±579)</td></tr><tr><td>PhTET3</td><td>Lys-pNA</td><td>0.5072 (±0.023)</td><td>35,760 (±1755)</td><td>70,501 (±4711)</td></tr></tbody></table>

Interestingly, the *k*<sub>cat</sub> values for PhTET2 and PhTET3 with their preferred substrates were comparable (approximately 35,000 s<sup>-1</sup>), whereas MjTET exhibited similar *k*<sub>cat</sub> values for both substrates tested (around 7100 s<sup>-1</sup>).

## Peptide digestion and HPLC assays

To further study the catalytic efficiency of MjTET, its activity was evaluated using three different synthetic peptides, each of them 15 amino acids in length. Additionally, its performance against each peptide was compared to that of PhTET2 and PhTET3, to evaluate how their differences in preferred monoacyl- substrates and kinetic constants translated to its enzymatic behavior toward complex peptide substrates.

The sequences of the peptides used were as follows: Pep1, LMLLNENRYTSWNSE (leucine-rich N-terminal, anticipated to be a better substrate for MjTET and PhTET2); Pep2, YFLMNENRYTSWNSE (aromatic-rich N-terminal, predicted to be cleavable by MjTET but not by the specialized TETs); and Pep3, KRRKEEDEYTSWNSE (lysine-rich N-terminal, expected to better suit PhTET3). For each enzymatic reaction, the resulting products were analyzed using HPLC, and the peaks corresponding to the main products were purified and sent for MS analysis to assess their identity (Figure 4).

From the results obtained it can be noticed that some amino acids not accepted as substrates in the monoacyl-pNA assays can be cleaved from the peptides by the TET enzymes. Pep1 was degraded by all three TETs, and MS analysis revealed that the shortest detectable product obtained was 9 amino acids in length, suggesting that all three enzymes processed the LMLLNE portion of the peptide, leaving NRYTSWNSE as the final product detected (appearing around 24 ml in the chromatogram). Interestingly, the HPLC chromatograms show that after 30 min, PhTET3 primarily produced this final product, indicating it was the most efficient one in degrading the peptide. PhTET2 also appears to outperform MjTET in degrading Pep1, showing a smaller relative presence of the peak corresponding to the initial peptide after 30 min of reaction. This indicates that MjTET left more of

the initial peptide unprocessed, when compared to both of the PhTET analogs.

In the case of Pep2, it was also unexpected to see that all three enzymes were able to process the peptide. Once again, PhTET3 was able to process further into the peptide after 30 min of incubation, showing the 9, 10, and 11 residue long products as the main peaks in the HPLC chromatogram. Also, after 30 min of incubation with PhTET3, the peak corresponding to the full-length initial peptide is completely absent from the chromatogram, suggesting the cleavage of the first residue (Tyr) was complete. Surprisingly, PhTET2 also outperformed MjTET in its activity towards this peptide, as evidenced by the faster appearance of the peak corresponding to the final product in the chromatograms of reactions carried with the former enzyme (already visible at 10 min). After 30 min, both reactions seemed to stop mostly at the 10/11 length peptide product (Appearing at around 32 ml in the chromatograms), meaning that the reaction proceeded through the YFLM portion of the peptide, slowing down at NENRYTSWNSE.

Finally, when looking at Pep3, tailored specifically as a better substrate for PhTET3, it can be readily observed from the HPLC analysis that PhTET3 processed the peptide further than the other enzymes, showing a peak in the chromatogram at about 24 ml that is absent from the chromatograms of the other two reactions in the times studied. This product was identified in MS analysis as TSWNSE. Furthermore, when comparing the chromatograms of the products of the reaction catalyzed by PhTET2 and MjTET, it can be noted from the larger number of peaks that the latter enzyme seems to have progressed further into the peptide than the former. This indicates that, as expected, PhTET3, followed by MjTET, were more efficient than PhTET2 in hydrolyzing Pep3.

## MjTET cryo-EM structure determination and comparison with the multiple TET system from Thermococcales

An atomic model of MjTET was built from cryo-EM data at a 3 Å resolution (Table 2). The monomer obtained has the typical structure of M42 aminopeptidases forming tetrahedral
<figure><img src="image_6.png" /><figcaption>**Figure 4.** HPLC analysis of the peptide degradation reactions with the three different TET aminopeptidases.
Reactions were incubated at 85 °C for 0, 10 and 30 min, in 50 mM buffer HEPES pH 8. The peptides were used at 0.5 mM and the enzyme concentration used was 3 µg/ml. Blue lines represent the chromatogram of the reactions performed with MjTET, green lines represent the chromatogram of the reactions performed with PhTET2, and the orange lines represent the chromatogram of the reactions performed with PhTET3. The chromatographic peaks of the main products were collected and sent for MS for analysis. The black arrows indicate the final products of the enzyme digestion.</figcaption></figure>

dodecamers (Figure 5A). The MjTET monomers can be divided in two α/β globular domains connected by two loops (residues 63–66 and 158–162). The bigger domain is the catalytic one formed by residues 1–66 and 158–349 and contains the N- and C-terminal ends. The smaller one is the dimerization domain (also called dimerization domain I) formed by residues 66–160. The catalytic domain contains a central eight-stranded β-sheet surrounded by seven α-helices. Additionally, a three-stranded antiparallel β-sheet (comprising residues 161–171 and 323–328) is positioned on the surface of the catalytic domain and is referred to as dimerization domain II (Figure 5B).

The dimerization domain I (residues 66–160) adopts a cylindrical β-barrel fold composed of six antiparallel β-strands and two α-helices, characteristic of PDZ-like domains. The overall architecture of this domain is highly conserved between MjTET and the PhTET enzymes. Within this domain, a loop spanning residues 112–125 is not resolved in the cryo-EM density map, suggesting substantial conformational flexibility. Consequently, its representation in the atomic model may not accurately reflect its native conformation.

The catalytic domain of MjTET contains the five conserved residues (H62, D175, E208, D230, and H321) responsible for coordinating the catalytic metal cations. The identity and spatial arrangement of these residues are highly conserved across M42 aminopeptidases. The

dodecahedral quaternary organization, characteristic of this enzyme family, confines the active sites within the central cavity of the particle. The catalytic domain also contains dimerization domain II, which likewise exhibits strong structural conservation among the available TET structures.

To gain insights into how the structure of these aminopeptidases can play a role in their substrate specificity and range, the structure of MjTET was compared to its four PhTET analogs, PhTET1, PhTET2, PhTET3 and PhTET4. The first three of them had structures already deposited in the PDB (PDB IDs: 2WYR, 1Y0R, 2WZN), while the latter was obtained as a prediction from the AlphaFold server. Structural alignment of the active site residues involved in metal ligand binding shows a high degree of conservation between all five enzymes (Figure 5C). When looking at the overall architecture of the monomers of the five enzymes, a similarly high degree of structural conservation can be observed (Figure 5D). The Cα RMSDs between MjTET and PhTET1–4 are 0.926, 0.644, 0.814, and 1.831 Å, respectively, indicating that the enzyme backbones are largely conserved. Nevertheless, notable differences in key structural features can be seen and are discussed in detail below.

## TET specificity pocket

Borisenko and Groll [14], characterized the specificity pocket (S1) in PhTET2, a region of the protein spanning residues Thr237, Ile238, Lys261, Glu291,
<table><caption>Table 2 Cryo-EM data collection, processing, model refinement, and validation statistics.</caption><tbody><tr><td>PDB code</td><td>9QR6</td></tr><tr><td>EMD code</td><td>EMD-53317</td></tr><tr><td colspan="2"><strong>Data collection and processing</strong></td></tr><tr><td>Magnification</td><td>45 k</td></tr><tr><td>Voltage (kV)</td><td>200</td></tr><tr><td>Electron exposure (e<sup>-</sup>/Å<sup>2</sup>)</td><td>50</td></tr><tr><td>Defocus range (μm)</td><td>-1.0 to -2.5</td></tr><tr><td>Pixel size (Å)</td><td>0.915</td></tr><tr><td>Symmetry imposed</td><td>T</td></tr><tr><td>Initial particle images</td><td>2,299,842</td></tr><tr><td>Final particles images</td><td>251,069</td></tr><tr><td>Map resolution (Å)</td><td>3.0</td></tr><tr><td>FSC threshold</td><td>0.143</td></tr><tr><td colspan="2"><strong>Refinement</strong></td></tr><tr><td>Initial model used (PDB code)</td><td>Alphafold prediction</td></tr><tr><td>Model resolution (Å)</td><td>3.2</td></tr><tr><td>FSC threshold</td><td>0.5</td></tr><tr><td>Map sharpening B factor (Å<sup>2</sup>)</td><td>-157.94</td></tr><tr><td colspan="2"><strong>Model composition</strong></td></tr><tr><td>Non-hydrogen atoms</td><td>32,133</td></tr><tr><td>B factors (Å<sup>2</sup>)</td><td>151.8 Å<sup>2</sup></td></tr><tr><td colspan="2"><strong>R.m.s deviations</strong></td></tr><tr><td>Bond lengths (Å)</td><td>0.008</td></tr><tr><td>Bond angles (°)</td><td>0.645</td></tr><tr><td colspan="2"><strong>Validation</strong></td></tr><tr><td>MolProbity score</td><td>1.99</td></tr><tr><td>Clashscore</td><td>8</td></tr><tr><td>Poor rotamers (%)</td><td>0.4%</td></tr><tr><td colspan="2"><strong>Ramachandran plot</strong></td></tr><tr><td>Favored (%)</td><td>97%</td></tr><tr><td>Allowed (%)</td><td>3%</td></tr><tr><td>Disallowed (%)</td><td>0%</td></tr></tbody></table>

Ile292, Leu293, Gly296 and Ile322, which was suggested to allocate the side chain of the residue that will be cleaved from the peptide substrate. To identify the residues in the other TETs that correspond to the S1 residues of PhTET2, the five structures were superimposed in PyMOL using the align command. While some of the S1 residues show clear conservation, some differences in their nature can be found between MjTET and its PhTET analogs.

In the S1 pocket, a highly conserved glycine residue appears to be crucial for substrate binding, as suggested by previous analyses of PhTET2 crystal structure [14]. Upstream of this glycine, three additional residues also involved in substrate binding show a variation pattern among TETs that could be related to the substrate specificity of each enzyme. This “S1 triad” is composed of E289, V290, and G291 in the case of MjTET; G278, V279, and T280 in PhTET1; E291, I292, and L293 in PhTET2; E289, P290, and S291 in PhTET3; and D279, V280, and F281 in PhTET4

(Supplementary Figure S2). Among the compared TET enzymes, this sequence begins with a negatively charged residue, except for the case of PhTET1, where a glycine replaces the acidic residue. The third residue in this sequence seems to be especially significant, as in PhTET1 and PhTET3, which preferentially bind amino acids with charged side-chains in their S1 pockets, this position is occupied by a polar amino acid (T280 in PhTET1 and S291 in PhTET3). In PhTET2, which preferentially degrades peptides with hydrophobic side-chains, this position is occupied by a hydrophobic leucine. These observations suggest that this “S1 triad” may play a role in determining the substrate preferences of TET enzymes. Also worth mentioning is the fact that in the case of MjTET, which has the broadest substrate range, the third residue in this sequence is Gly291. In contrast, in PhTET4, which exhibits a narrow preference for peptides with the smallest possible N-terminal side chain (glycine), this position is occupied by a bulkier residue, Phe281.

## Electrostatic surface analysis

Examination of the electrostatic surface inside the particles (Figure 6) reveals patterns that may explain the substrate preference diversity among TETs. To visualize the interior, hexameric models were generated in PyMOL, and electrostatic surfaces were calculated at a common potential level of ±10. This analysis showed that MjTET exhibits a heterogeneous charge distribution within the internal cavity, characterized by predominantly negative regions around the active site, surrounded by uncharged patches and smaller positive areas contributed mainly by residues in the dimerization domain and by K212. Interestingly, the positive charge contributed by this residue is absent from PhTET2 and PhTET3 but present in PhTET1 (K209).

Unlike the other TETs in this comparison, on the inside of the particle, PhTET1 exhibits large positive patches around the active sites, contributed mainly by residues K303, K240, K212, K209, R89, and R81. These patches are accompanied by a smaller, localized negative surface containing the catalytic metal binding residues. In PhTET2, the negative pocket containing the catalytic residues is surrounded by mostly uncharged regions, similarly to MjTET. PhTET3 exhibits a largely negative surface on the inside the particle, owing to the presence of E99, E215 and E289. Inside the PhTET4 oligomeric particle, the notably localized negative charges from the active site residues are surrounded by mostly uncharged patches. This shows that in each of these TET aminopeptidases, the surface charges inside the particles are consistent with what could be expected given their substrate selectivity.
<figure><img src="image_7.png" /><figcaption>**Figure 5.** Structural analysis of MjTET. (A) Atomic model of the dodecameric MjTET particle. The protein is shown from a vertex, with each monomer shown in a different color. (B) Representation of an MjTET dimer. The catalytic domain is shown in cyan and the dimerization domain in pink. The flexible loop of the dimerization domain is depicted in white. The small three-stranded antiparallel β-sheet located in the catalytic domain but implicated in oligomerization (dimerization domain II) is highlighted in blue. (C) Superposition of active-site residues involved in metal coordination in MjTET and its four PhTET homologs. The MjTET cryo-EM structure was aligned in PyMOL with the crystal structures of PhTET1–3 (PDB IDs: 1y0r, 2wzn, 2wyr) and the AlphaFold3-predicted model of PhTET4. Active-site residues show a high degree of positional conservation across all five structures. (D) Comparison of monomers from five TETs. Catalytic domains are colored cyan, magenta, green, orange, and wheat for MjTET, PhTET1, PhTET2, PhTET3, and PhTET4, respectively; dimerization domains are shown in violet. Residues involved in metal binding are depicted in stick representation, while S1 pocket residues are highlighted in yellow. The flexible loop of the dimerization domain is shown in white for MjTET, and as a dotted line for PhTET1–3.</figcaption></figure>

## Size of the catalytic chamber

From the analysis of the electrostatic surface, differences in the accessibility of the active site could be observed among the TETs compared. Notably, the catalytic pocket in PhTET4 appears to be deeply buried within the structure in the surface representation of the protein. Conversely, MjTET represent an opposite case, where the overall architecture of the catalytic chamber created by the three monomers joining at the apexes allows for a more exposed active site. Indeed, when analyzing the side chains around the variable S1 triad and the conserved glycine, it can be seen that in MjTET, specifically positioned glycines (G256, G291, G293) allow for a wider S1 pocket (Supplementary Figure S3). In contrast, in PhTET4, bulkier side chains from residues Y257, F281, Y283, and Y284 contribute to forming the narrower S1 pocket of all the TETs compared (Figure 7). PhTET1 follows a similar trend, with some markedly bulkier side chains in comparison

to their equivalent S1 residues in MjTET. In PhTET1, the presence of F229, R248, and T280 (positionally equivalent to T232, G256, and G291 in MjTET, respectively) impose steric hindrances absent in MjTET. While PhTET2 shows the strongest structural conservation in relation to MjTET, the presence of L293 (positionally equivalent to G291 in MjTET) also has a similarly narrowing effect. PhTET3 seems to deviate slightly from this trend, as the overall architecture of the loops in its S1 pocket forms a wide catalytic chamber. Still, the presence of D262 (equivalent to G256 in MjTET), and F241 (equivalent to I233 in MjTET), contribute steric bulk in the S1 pocket.

## Variable loop

Starting three residues after an active-site Asp that coordinates one of the metal ligands, there is a loop exposed towards the wide pore on the faces of the dodecameric particle (Figure 8A).
<figure><img src="image_8.png" /><figcaption>**Figure 6.** Internal electrostatic surfaces of MjTET and its four PhTET homologs. The MjTET cryo-EM structure (PDB ID: 9qr6) was compared with the crystal structures of PhTET1 (PDB ID: 2wyr), PhTET2 (PDB ID: 1y0r), and PhTET3 (PDB ID: 2wzn), as well as the predicted PhTET4 model generated with AlphaFold3. Representations show the interior surface of the particles. Active sites are indicated with arrows. Electrostatic potentials were calculated with the APBS plugin in PyMOL and are displayed at ±10 kT/e.</figcaption></figure>

This region shows prominent structural and sequence variation across different TETs. In particular, its length varies among the enzymes studied: it is 19 residues long in PhTET2 and MjTET, 17 residues long in PhTET3, and 14 and 10 residues long in PhTET1 and PhTET4, respectively. This variation pattern seems to reflect the substrate specificity of each enzyme, with the longer loop length corresponding to MjTET, the enzyme with the broader substrate specificity, and the shorter one corresponding to PhTET4, which has the narrower substrate specificity. These variable loops feature a highly conserved LGKG sequence present in four of the TETs studied, but absent in the highly specialized PhTET4.

With this in mind, we produced two MjTET mutants with a shortened variable loop, to evaluate if this would reduce its substrate specificity using monoacyl assays: MjTET-ΔLoop, which has this region completely deleted, and MjTET-LoopT4, which has this loop swapped for the one from PhTET4 (Figure 8B).

In terms of specific activity, both mutants showed a reduction when compared to MjTET-wt. Specific activity against pNA-Leucine was 25 (±0.79) U/mg for MjTET, 4.2 (±0.12) U/mg for MjTET-ΔLoop, and 11 (±0.93) U/mg for MjTET-LoopT4. Interestingly, although specific activity was lower in mutants, their activity spectrum was found to be significantly affected, as shown in figure 8C.

Indeed, while the wildtype MjTET (MjTET-wt) has comparable activity against glutamine, methionine and leucine, MjTET-ΔLoop shows a marked specificity towards leucine, with the activity against other residues greatly reduced (Figure 8C). The relative activities of MjTET-ΔLoop are close to 20% towards threonine and methionine, and around 15% towards serine and glutamine. Relative activity against lysine is about 10%, whereas in the case of MjTET-wt it's around 60%. Relative activities against other monoacyl-substrates were in the range of 5% and below.

In the case of MjTET-LoopT4, we observed a similar tendency but slightly less pronounced. This mutant also shows clear preference for leucine,
<figure><img src="image_9.png" /><figcaption>**Figure 7.** Structural alignment of the S1 pocket. Atomic models of PhTET1 (in magenta, panel A), PhTET2 (in green, panel B), PhTET3 (in orange, panel C), and PhTET4 (in wheat, panel D) are superimposed on a surface representation of MjTET (cyan). Residues forming the S1 pocket are shown in yellow. The bulkier side chains (indicated by arrows) of the PhTETs extend into regions that are solvent-accessible within the wider cavity of MjTET.</figcaption></figure>

while maintaining close to 50% relative activity towards methionine. It shows over 20% relative activity towards glutamine, and comparable activities in the range of 10–15% towards serine, tryptophan, isoleucine, lysine and threonine. For all the other monoacyl substrates tried, relative activity was either less than 10% or undetectable.

These results indicate that the variable loop within the catalytic domain plays a role in substrate discrimination, as both its deletion and shortening increased selectivity towards leucine in the mutants tested.

Discussion

Genomic surveys and functional characterizations indicate that the number of TET proteins in prokaryotic cells can range from one to as many as four distinct versions. In the deep-sea hyperthermophilic archaeon *P. horikoshii*, four different TET complexes with varying substrate preferences have been identified [6,8,9,13]. By contrast, *M. jannaschii*, another hyperthermophilic

archaeon from a similar natural environment, contains only one gene encoding a putative M42 family TET. In this study, we demonstrated that the corresponding recombinant protein, referred to as MjTET, is indeed a hyperthermophilic TET aminopeptidase. The enzyme forms dodecamers of a molecular mass around 460 kDa, similar to that of analogous TET proteins from *P. horikoshii* and *P. furiosus* [5,7], and shows maximal activity at 85 °C and slightly alkaline pH 8.

Similar to other TET enzymes, MjTET requires a metal cofactor for aminopeptidase activity, showing maximal activity with 0.1 mM Co²⁺. Higher Co²⁺ concentrations inhibited the enzyme, as has been reported for other TET aminopeptidases [15]. In the case of PfTET3, produced by *P. furiosus*, cobalt has been shown to be more important as an activator at high temperatures, where it is hypothesized to increase enzyme stability by adding rigidity. At lower temperatures (<50 °C), however, cobalt has an inhibitory effect on its aminopeptidase activity [15]. This was not observed with MjTET, which showed activation by
<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

**Figure 8.** Structural and functional analysis of the variable loop in TETs. (A) Structural and sequence comparison of the variable loop. Monomers of the five TETs are superimposed (MjTET, cyan; PhTET1, magenta; PhTET2, green; PhTET3, orange; PhTET4, yellow). The variable loop region is circled, and the corresponding sequence alignment is shown below. Red boxes highlight the loop region; the conserved Asp residue three positions upstream, coordinating one of the metal centers, is marked with a star. (B) AlphaFold predictions of the structures of the two MjTET loop mutants: the loop-deletion mutant (MjTET-Δloop, cyan) and the PhTET4 loop-swap mutant (MjTET-LoopT4, pink). (C) Substrate specificity of the two MjTET loop mutants. Relative activity toward monoacyl substrates was measured; both mutants show increased selectivity for leucine compared with MjTET-wt.

cobalt at both 75 °C and 50 °C, indicating that cobalt is important for activity across the entire temperature range.

The monoacyl substrate screening revealed that the enzyme is a broad-spectrum aminopeptidase, with higher activities toward glutamine, methionine and leucine, and a strong secondary activity towards lysine. MjTET is capable of cleaving hydrophobic, polar, aromatic and positively charged residues, and can therefore be classified as a *generalist* TET, as opposed to the *specialized* PhTETs. In general, M42 aminopeptidases have displayed poor amidolytic activity against aromatic substrates such as phenylalanine-pNA and tyrosine-pNA, but this is not the case for MjTET. Interestingly, while the TETs from *P. horikoshii* have complementary substrates specificities, spanning hydrophobic, acid and basic residues, the results from our monoacyl substrate assays shows that the generalist MjTET is not able to degrade negatively charged amino acids as substrates. In a recently

published study, an extensive analysis of archaeal TET sequences was conducted, along with the biochemical characterization of representative members from the different families identified [16]. The results show that family 11, to which MjTET belongs, is in fact the most widely represented TET family in archaea, suggesting that broad substrate preference is the norm among archaeal TET enzymes.

While differences in substrate preference were more pronounced in the monoacyl hydrolysis assays, the three TETs tested performed better than anticipated when acting on complex peptides, successfully cleaving residues that were not detected as substrates in the monoacyl assays. This suggests that, despite the preference for specific N-terminal residues indicated by the monoacyl-substrate assays, substrate discrimination and functional specialization among archaeal TETs may be more nuanced than previously speculated. This could imply that, although M42 aminopeptidases can evolve to fine-
tune their preference toward particular amino acid types, in the context of peptides they retain the ability to hydrolyze diverse substrates with varying efficiencies. This observation led us to explore how the different catalytic efficiencies observed among archaeal TETs might relate to their physiology and metabolic roles.

Interestingly, both PhTET3 and PhTET2 exhibited higher overall activity in peptide digestion compared to MjTET. The catalytic efficiencies measured for their preferred pNA substrates were also greater than those calculated for MjTET. These findings indicate that substrate specialization in PhTET enzymes is associated with enhanced catalytic efficiency. Accordingly, the presence of four specialized TET aminopeptidases in *P. horikoshii* likely provides a more efficient amidolytic system than the single, generalist TET present in *M. jannaschii*, reflecting a greater demand for peptide hydrolysis in the former organism.

Indeed, unlike *P. horikoshii*, a heterotroph that relies on environmental biomolecules as carbon sources and uses peptide fermentation for energy production [17], *M. jannaschii* is an autotroph that supports its metabolism through methanogenesis [18]. This difference in metabolic strategies may explain the presence of a single generalist TET in *M. jannaschii* compared to the system of multiple specialized TETs found in Thermococcales. The *Pyrococcus* genus may have evolved a suite of enzymes with complementary activities to efficiently process peptides into amino acids, which are critical for Thermococcales, as they use amino acids for both protein synthesis and energy production. In contrast, the single generalist TET in *M. jannaschii* likely serves functions unrelated to its energetic needs, such as amino acid recycling, peptide homeostasis or N-terminal processing.

When analyzed together, MjTET and the four PhTETs display a spectrum of substrate specificities, with MjTET being the most promiscuous of these five enzymes. PhTET2 and PhTET3 exhibit slightly broader substrate preferences but show a clear selectivity against certain amino acid types. PhTET1 has activity limited to peptides with an N-terminal Asp or Glu. Finally, the narrowest specificity is observed in PhTET4, which acts exclusively on N-terminal glycine. In an attempt to correlate these biochemical differences to variations observed in their structures, we compared the atomic models of these five enzymes.

First analyses reveal that the architecture and identity of the active site residues remain conserved across all TETs studied to date, linking them to other binuclear metalloaminopeptidases such as *Aeromonas proteolytica* aminopeptidase and bovine leucine aminopeptidase, with which they share a common catalytic mechanism [14].

This active site conservation implies that differences correlated to substrate specificity should be found somewhere else in the structure and are related to substrate binding and not catalysis. This was also reflected by the differences in catalytic constants obtained for the two substrates assayed with MjTET: The $k_{cat}$ was similar for both, while $K_M$ showed important variability, indicating that the measurable differences in activity are explained by differences in binding affinity. Following this reasoning, important differences were observed in the S1 pocket, where the side chain of the N-terminal residue to be cleaved interacts with the protein. We identified a triad of S1 residues that seem to vary in a manner consistent with the substrate specificities of each enzyme. In four of the compared TET enzymes, this triad begins with a negatively charged residue. However, in the case of PhTET1, a glycine replaces the acidic residue, which reflects the requirement to accommodate a negatively charged side chain in the S1 pocket of this specialized TET. Furthermore, the third residue of this “S1 triad” also follows a pattern that may be relevant to substrate preferences. In PhTET1 and PhTET3, which need to accommodate charged side chains in their S1 pockets, this position is occupied by a polar residue (threonine and serine, respectively). In PhTET2, which binds hydrophobic side chains, this position is occupied by a hydrophobic residue, leucine. In the generalist MjTET, this position is taken by a glycine, which, due to its lack of a large side chain, may allow the enzyme to accommodate a broader range of substrates. Consistently, in PhTET4, this position is occupied by a bulky aromatic residue, phenylalanine. These observations strongly suggest that variability in this “S1 triad” reflects functional adaptations that enable different substrate specificities.

When examining the electrostatic surface of the dodecamers, further patterns that reflect the biochemical differences between the five TETs compared emerge. While all the enzymes analyzed show a negatively charged active site, corresponding to the catalytic residues and the metal cofactor binding sites, variations in the surrounding charge distribution align with substrate specificity: MjTET features a broadly negative active site surrounded by hydrophobic patches, which may explain its affinity for both polar residues (e.g., glutamine) and hydrophobic residues (e.g., methionine and leucine). In contrast, PhTET2 and PhTET4 exhibit highly localized negative charges in the active site, surrounded by uncharged regions, consistent with their preference for hydrophobic substrates. PhTET3, on the other hand, presents a largely negative surface around the catalytic residues, aligning with its preference for positively charged substrates. Together, these observations suggest that surface charge distribution in the interior of
the dodecameric particles is also relevant in
determining TET substrate preference.

A third structural element linked to substrate specificity is the volume of the catalytic chamber and accessibility of the active site. At one end of the spectrum, PhTET4 has a compact catalytic chamber, with bulky side chains hindering access to the active site, consistent with its tight preference for peptides with an N-terminal glycine. On the opposite end, MjTET, which has the broadest substrate specificity among the five enzymes, also shows the largest catalytic pocket. While PhTET3 deviates from this trend, possessing a wide catalytic chamber, its strong preference for basic substrates is likely driven by the charge restriction introduced by the highly negatively charged interior surface of the dodecameric particle. These findings suggest that substrate selectivity in TET aminopeptidases results from an interplay of multiple structural factors.

Another feature of this enzyme family that appears to influence substrate specificity is the variable-length loop located between the active site residues and the face of each monomer oriented toward the wide pore of the tetrahedral dodecamer. If substrates enter the particle through this pore, the incoming peptide would encounter the loop before reaching the S1 pocket and the active site. The length and flexibility of this loop may affect substrate binding, with shorter and less flexible loops potentially correlating with narrower substrate specificity. Although a direct comparison of the residue-averaged B-Factors cannot be made between the structures, as they were obtained using different methods, variations in the values along the flexible loop may indicate a higher flexibility of this region in MjTET when compared to the PhTET1-3 (Supplementary Figure S4). Conversely, longer loops could facilitate more stabilizing interactions between the enzyme and the substrate, potentially enhancing specificity.

To test this hypothesis, we engineered two MjTET mutants with shortened loops. Both MjTET-ΔLoop and MjTET-LoopT4 exhibited a reduced substrate range compared to wild-type MjTET, and an increased preference for leucine. In both mutants, activity toward positively charged residues (lysine and arginine) was diminished, while selectivity shifted toward hydrophobic and uncharged polar residues. Although overall specific activity was affected, indicating that mutating this loop affects catalysis, the relative specificity for different monoacyl substrates was significantly altered. These findings demonstrate that this loop plays a key role in determining substrate specificity. To our knowledge, this is the first time its functional importance has been identified in TET aminopeptidases.

In summary, our analysis of MjTET and the four PhTET structures demonstrates that, despite a highly conserved overall architecture, several structural differences likely contribute to their distinct substrate specificities. These differences include variations in the size and nature of amino acid side chains within the S1 pocket, the distribution of surface charges, the dimensions of the catalytic chamber, and specific loops within the catalytic domain. Collectively, these findings underscore that substrate specificity in TET aminopeptidases is not determined by a single structural feature, but by a combination of tertiary structure, oligomeric assembly, and electrostatic properties. Additionally, it is noteworthy that PhTET4 exhibits a unique preference for nickel as its catalytic metal ion [6], in contrast to other TETs described to date, which use cobalt instead. This observation raises the possibility that metal ion selection may also influence substrate specificity, a hypothesis that merits further investigation. Together, these factors contribute to the distinct and sometimes complementary enzymatic activities observed across TET aminopeptidases.

Our findings indicate that the structural and biochemical adaptations of TET aminopeptidases reflect the metabolic needs and ecological contexts of the hyperthermophilic archaea that produce them. They also highlight the role of compartmentalization within these dodecameric complexes, where pores, cavities, and charged microenvironments shape enzyme-substrate interactions. A deeper understanding of the determinants of substrate specificity and catalysis in these enzymes opens the door to rational engineering for biotechnological applications. These range from the pharmaceutical industry, where selective peptide modification is valuable, to biomass processing for enriched hydrolysates used in human nutrition, livestock feed, and aquaculture [19–21]. However, given the complex interplay of factors governing substrate specificity in TET enzymes, such engineering may pose significant challenges. The present study represents a step toward overcoming these challenges and harnessing the potential of this unique class of enzymes.

# Materials and Methods

## Protein expression and purification

The genes encoding MjTET and its two mutants MjTET-ΔLoop and MjTET-LoopT4 were synthesized and cloned by GeneCust Europe in pET-41c. The proteins were then produced and purified using protocols based on those developed for the hyperthermophilic TET from *P. horikoshii* [6]. We used *Escherichia coli* ArcticExpress (DE3)-RIL strain (Agilent Technologies) for the
over-expression of the recombinant protein. The cells were grown in 1 L of lysogeny broth (LB) medium containing gentamycin and kanamycin at 50 µg/ml. Then the cells were incubated with shaking at 37 °C until the OD₅₀₀ reached 0.6–1.0, at this stage the induction was carried out by adding isopropyl β-D-thiogalactopyranoside at final concentration of 0.1 mM and proteins were overexpressed during 20h at 16 °C. The induced cells were harvested by centrifugation, and the pellet was then conserved at –80 °C until use. The pellet was suspended in 50 ml of 50 mM Tris–HCl, 200 mM NaCl and 0.1% Triton X-100, pH 7.5, supplemented with 12.5 mg of lysozyme (Euromedex), 2.5 mg of DNase I grade II, 10 mg of RNase, 50 mg of Pefabloc SC (Roche), 500 µl of 2 M MgSO₄. The cell disruption was achieved by sonication in a Branson sonifier at 30 W for 10 cycles on/off of 30 sec each, at 4 °C. The crude extracts were heated at 85 °C for 20 min and later clarified by centrifugation (JA20 rotor) at 12000 rpm for 1 h at 4 °C. The supernatant was diluted two times with 20 mM Tris–HCl pH 7.5 and loaded on anion exchange column (Resource Q) (GE Healthcare Life Sciences). The protein was eluted with a linear NaCl gradient from 190–340 mM in 20 mM Tris–HCl pH 7.5. Size exclusion chromatography was done on Superose 6 column or Superdex 200 column (GE Healthcare Life Sciences) previously equilibrated with 20 mM Tris–HCl, 200 mM NaCl, pH 7.5. The fractions containing the protein according to SDS-PAGE were pooled and concentrated using Amicon cell (Millipore) with a molecular mass cutoff of 30 kDa and kept at 4 °C. PhTET2 and PhTET3 were purified as described in [7,13].

## Negative Staining electron microscopy

MjTET samples, at approximately 0.1 mg/mL in 20 mM Tris–HCl and 200 mM NaCl, pH 7.5, were adsorbed to the clean side of a carbon film on mica, stained with 2% sodium silicotungstate pH 7.5, and transferred to a 400-mesh copper grid. The images were taken on a Tecnai 12 LaB6 electron microscope at 120 kV accelerating voltage using CCD camera Gatan Orius 1000.

## Enzymatic assays

The activity of MjTET on synthetic chromogenic (aminoacyl-pNA) and fluorogenic (aminoacyl-AMC) substrates was assayed as follows. Reactions were initiated by adding 3 µg/mL enzyme to a pre-warmed mixture containing 5 mM substrate in 50 mM HEPES (pH 8.0), 150 mM KCl, and 0.1 mM CoCl₂, in a total volume of 300 µL. Incubations were carried out at 80 °C for 5 min. This temperature was chosen because, above 80 °C, some substrates become unstable and undergo significant nonenzymatic degradation. Release of pNA was quantified by absorbance at 405 nm using a JASCO V-630

spectrophotometer, and release of AMC was quantified by fluorescence (excitation 360 nm; emission 440 nm) using a JASCO FP-8500 spectrofluorometer. Each assay was performed in triplicate with two enzyme blanks as controls. Specific activity was calculated as described by Spungin and Blumberg [22]. To calculate the catalytic constants, the rate of the reaction was calculated for concentrations of aminoacyl-pNA substrates ranging from 0.025 mM to 2.5 mM. Reactions were incubated at 75 °C instead of 80 °C to facilitate measurement of the initial reaction rate and improve the accuracy of the kinetic analysis. Kinetic parameters were determined by nonlinear least-squares fitting of the experimental data.

## Effect of temperature, pH and metal cations on MjTET activity

All assays were performed in the presence of 150 mM KCl, 0.1 mM CoCl₂, 4 µg/mL MjTET, and 5 mM Leu-pNA. Reactions were incubated at the desired temperature for 5 min, and pNA release was monitored at 405 nm on a JASCO V-630 spectrophotometer.

The effect of pH on MjTET activity was tested from pH 6 to 10, using 50 mM HEPES (pH 6–7.5) and 50 mM CHES (pH 8.2–10). Buffers were adjusted at 75 °C, and the assays were carried out at this temperature. Temperature dependence was then assessed from 20 to 90 °C in 50 mM CHES, pH 8.2. Metal dependence was evaluated by replacing CoCl₂ with 0.1 mM XCl₂ salts of the indicated metals, using 50 mM CHES at pH 8.2. All reactions were carried out as described above.

## TET degradation of synthetic peptides

The capacity of MjTET, PhTET2 and PhTET3 to degrade longer substrates was assessed using three synthetic peptides: Pep1 (LMLLNENRY-TSWNSE), Pep2 (YFLMNENRYTSWNSE) and Pep3, (KRRKEEDEYTSWNSE). All enzymes used at 0.5 mg/ml and peptides were added at a final concentration of 0.5 mM in the reaction media. The reaction was incubated at 85 °C for different times and then stopped with 1 vol of 10% acetic acid and immediately placing the containing tubes in ice. Aliquots of 80 µL were then removed and added to 220 µL of 2% acetonitrile (ACN), 0.1% trifluoroacetic acid (TFA). Proteins were removed by centrifugation at 13,000 rpm during 15 min. 100 µL of the supernatant were retrieved and injected on Nova-Pak C18 column, (4 µm, 3.9 × 300 mm, (Waters)) in a HPLC system (PerkinElmer), equilibrated with 2% ACN, 0.1% TFA. The elution of peptide products was achieved with a linear ACN gradient (2–30%) and followed by measuring the absorbance at 214 nm. Chromatographic runs were carried out at room temperature.
## MS analysis of the peptide digestion products

The main peaks from the HPLC analysis of peptide digestion reactions were collected during chromatographic elution and analyzed by mass spectrometry. Isolated peaks were dried in a Speed-Vac, then resuspended in 100 µL of 0.1% formic acid. 1 µL was injected onto a pre-column, followed by separation on an Aurora C18 Ion-Optic column (25 cm, 1.7 µm beads) using a 25-min acetonitrile gradient. MS and HCD-MS/MS analyses were performed on an Ascend Orbitrap system. Fragmentation was carried out on precursor ions with charge states from +1 to +5. The mass range analyzed was m/z 300–1600, with a threshold of 5 × 10⁶ to exclude background ions in data-dependent acquisition.

## Cryo-EM grids preparation, data collection and image analysis

3.5 µL of sample at 1 mg/mL was applied on freshly glow discharged grids (Ultra-Au 1.2/1.3 grids; Quantifoil) at 20 °C and 100% humidity using a Vitrobot Mark IV (Thermo Fisher). The grids were glow discharged in air for 45 s at 30 mA (Emitech Glow Discharge). The excess of liquid was blotted 5.5 s at blot force 0 and 0.5 s drain time before vitrification in liquid ethane.

Automated data collection was performed on a 200 kV Glacios cryo-TEM microscope (Thermo Fischer Scientific) equipped with a K2 direct electron detector (Gatan) using SerialEM [23]. Coma and astigmatism corrections were also performed using SerialEM. Movies of 40 frames were recorded in counting mode at a 45,000× magnification giving a pixel size of 0.915 Å with defocus ranging from -1.0 to -2.5 µm using a multi-shot scheme (3 × 3 grids of holes without moving the stage). Total exposure dose per movie was 50 e⁻/Å² and total number of images was 5898.

Movie drift correction and CTF determination were performed with Relion [24]. 5706 images of good quality were kept for the picking. More than 2,000,000 particles were automatically selected into 300 × 300 pixels boxes. The particles were submitted to 2D classification. After extensive selection and generation of an initial model imposing Tetrahedral symmetry, 3D refinement and post processing generated a final reconstruction including 251,069 particles with a resolution of 3.0 Å (FSC = 0.143). A graphical flowchart summarizing the workflow can be seen in **supplementary Figure S5**.

## Structure refinement and analysis

The resulting EM map was used for model building in both Phenix and Coot software, using an AlphaFold prediction as starting model. The quality of the obtained model was assessed

through MolProbity and the PDB validation platform for successive refinement steps. For structure visualization and comparison between available TET atomic models, PyMOL and ChimeraX were used [25,26]. The final model was deposited into the PDB (PDB ID 9QR6 and EMD-53317).

## CRediT authorship contribution statement

**Joaquin Atalah:** Writing – review & editing, Writing – original draft, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Hind Basbous:** Methodology, Investigation. **Gregory Effantin:** Formal analysis. **Sylvie Kieffer-Jaquinod:** Formal analysis. **Guy Schoehn:** Supervision, Methodology, Formal analysis. **Eric Girard:** Project administration, Conceptualization. **Bruno Franzetti:** Writing – review & editing, Supervision, Conceptualization.

## DATA AVAILABILITY

Data will be made available on request.

## DECLARATION OF COMPETING INTEREST

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgments

This project received funding from GRAL, a program from the Chemistry Biology Health (CBH) Graduate School of University Grenoble Alpes (ANR-17-EURE-0003), and was also partially financed by ANID through Becas Chile scholarship program. The work was made possible through access to the platforms of the Grenoble Instruct-ERIC centre (ISBG; UAR 3518 CNRS-CEA-UGA-EMBL), within the Grenoble Partnership for Structural Biology (PSB), supported by FRISBI (ANR-10-INBS-0005-02) and GRAL. The IBS-ISBG EM facility is supported by the Auvergne-Rhône-Alpes Region, the Fondation Recherche Medicale (FRM), the fonds FEDER and the GIS-Infrastructures en Biologie Sante et Agronomie (IBISA). We thank Daphna Fenel for the negative staining experiment. In addition, MS experiments were supported by Agence Nationale de la Recherche under ProFI (Proteomics French Infrastructure, ANR-10-INBS-08).
## Appendix A. Supplementary material

Supplementary material to this article can be found online at https://doi.org/10.1016/j.jmb.2025.169596.

Received 29 September 2025;
Accepted 11 December 2025;
Available online 17 December 2025

**Keywords:**
peptidases;
hyperthermophiles;
TET;
M42;
archaea

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