# Widespread deployment of the human CD38 ADP-ribosyl cyclase fold in antibacterial and anti-eukaryotic polymorphic toxins

Julius Martinkus, Laurent Terradot, Dukas Jurènas, Eric Cascales

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Julius Martinkus, Laurent Terradot, Dukas Jurènas, Eric Cascales. Widespread deployment of the human CD38 ADP-ribosyl cyclase fold in antibacterial and anti-eukaryotic polymorphic toxins. Journal of Biological Chemistry, 2025, pp.110775. 10.1016/j.jbc.2025.110775. hal-05335759

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Widespread deployment of the human CD38 ADP-ribosyl cyclase fold in
antibacterial and anti-eukaryotic polymorphic toxins

Julius Martinkus¹, Laurent Terradot², Dukas Jurènas³,⁴*, Eric Cascales¹,*

Keywords: type VI secretion, Polymorphic toxin, NAD⁺, ADP ribosyl cyclase, CD38

¹ Laboratoire d'Ingénierie des Systèmes Macromoléculaires (LISM), Aix-Marseille Université,
CNRS, UMR 7255, Marseille, France.

² Laboratory of Molecular Microbiology and Structural Biochemistry, Institut de Biologie et
Chimie des Protéines (IBCP), Université Claude Bernard, CNRS, UMR 5086, Lyon, France.

³ Bacterial Genetics and Physiology, Faculté des Sciences, Université Libre de Bruxelles
(ULB), Gosselies, Belgium

⁴ WEL Research Institute, Wavre, Belgium.

* To whom correspondence should be addressed: Dukas Jurènas (dukas.jurenas@ulb.be), Eric Cascales (cascales@imm.cnrs.fr)

Running title: NAD/NADP depletion by CD38-like NAD glycohydrolase toxins
31 ABSTRACT

32 Bacterial polymorphic toxins are modular weapons that mediate inter-microbial
33 competition and host interactions by delivering diverse cytotoxic domains through
34 specialized secretion systems. Here, we identify and characterize a novel toxin domain in
35 *Pantoea ananatis* that displays remarkable structural and functional conservation with
36 the human enzyme CD38. This bacterial toxin, fused to a type VI secretion system (T6SS)
37 PAAR domain, harbors a C-terminal ADP-ribosyl cyclase (ARC) domain that hydrolyzes
38 NAD+ and NADP+ *in vitro* and *in vivo*, leading to growth inhibition in both bacterial and
39 eukaryotic cells. The 1.6-Å resolution structure of ARC reveals that it adopts a globular
40 fold nearly identical to the human CD38 ADP ribosyl cyclase, with key catalytic residues
41 conserved. Comparative genomics reveals that CD38-like ARC domains are widespread
42 in bacteria, fused to diverse delivery modules including T6SS, T7SS, and CDI systems.
43 Functional assays demonstrate that these domains act as NAD-depleting toxins, with
44 cross-immunity observed between non-cognate toxin-immunity pairs. Taken together,
45 our findings identify a bacterial NAD+ hydrolase fold with strong similarity to human
46 CD38 and define a novel class of metabolic toxins, expanding the functional scope of
47 polymorphic effectors and illustrating how enzymes can be co-opted for microbial
48 warfare.
49

49
50 INTRODUCTION

Bacteria live in densely populated, competitive environments where access to nutrients and space is limited (Little et al., 2008; Hibbing et al., 2010; Ghoul and Mitri, 2016; Garcia-Bayona and Comstock, 2018; Peterson et al., 2020). To thrive, many species have evolved potent weapons to inhibit or eliminate their rivals (Ruhe et al., 2013; Russell et al., 2014; Jamet and Nassif, 2015; Chassaing and Cascales, 2018; Cherrak et al., 2019; Klein et al., 2020; Kern et al., 2021; Cuthbert et al., 2022; Boardman et al., 2023). Among the most versatile of these weapons are polymorphic toxins (PT), which are modular, multidomain proteins that are delivered into neighboring cells to sabotage essential processes and cellular functions (Zhang et al., 2012; Jamet and Nassif, 2015; Ruhe et al., 2020). These toxins are often secreted through sophisticated nanomachines, such as the type IV (T4SS), type V (T5SS), type VI (T6SS) or type VII (T7SS) secretion systems (Shen et al., 2012; Souza et al., 2015; Willett et al., 2015; Bayer-Santos et al., 2019; Klein et al., 2020; Cherrak et al., 2019; Hernandez et al., 2020; Jurenas and Journet, 2021; Boardman et al., 2023; Lin, 2024), and are typically accompanied by immunity proteins that protect the producing bacterium from self-intoxication (Benz and Meinhardt, 2014; Jana and Salomon, 2019). The diversity and adaptability of polymorphic toxins have made them central players in interbacterial warfare, microbial community structuring, and even host interactions (Ruhe et al., 2020).

A defining feature of polymorphic toxins is their modularity. These proteins share a conserved N-terminal trafficking domain fused to highly variable C-terminal "effector" domains, which confer toxic activity (Zhang et al., 2012; Jamet and Nassif, 2015; Ruhe et al., 2020). Over evolutionary time, this modularity has enabled the shuffling and specialization of toxin domains, resulting in a vast repertoire of enzymatic activities including nucleases, peptidases, lipases, or NAD-targeting enzymes (Zhang et al., 2012). Increasingly, it has become clear that bacteria do
74 not solely evolve new toxic domains de novo; they also select, co-opt and repurpose host-like proteins for bacterial warfare (Ruhe et al., 2020; Aravind et al., 2024; Anshul and Kumari, 2025).
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76

77 One of the most intriguing examples is toxin mimicry, i.e. the occurrence of eukaryotic-like domains in bacterial toxins, which can closely resemble host enzymes in structure and function (Chen and Xia, 2021; Martyn et al., 2022; Anshul and Kumari, 2025). Such similarities may arise from shared biochemical constraints, convergent evolution or ancient common ancestry. 78
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81 This results in bacterial effectors that perform activities analogous to those of host proteins and used in competition, colonization, or immune evasion (Chen and Xia, 2021; Martyn et al., 2022).
82
83 Notably, several bacterial toxins appear to mimic or structurally resemble eukaryotic proteins involved in key cellular processes, such as actin polymerization, cell signaling, and nucleotide metabolism (Stebbins and Galan, 2001; Mak and Thurston, 2021, Chen and Xia, 2021; Anshul and Kumari, 2025). 84
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87 In this study, we describe the discovery of a novel toxin domain in *Pantoea ananatis* that exemplifies this strategy of molecular hijacking. The toxin, encoded within a T6SS vgrG/hcp/paar island, harbors an N-terminal Proline-Alanine-Alanine-Arginine (PAAR) trafficking domain, putative transmembrane helices and a C-terminal domain with strong structural and functional similarity to the human enzyme CD38 (Cluster of Differentiation 38), a multifunctional ADP-ribosyl cyclase (ARC) involved in immune cell activation and regulation of calcium intracellular levels and signaling (De Flora et al., 1997; Schuber and Lund, 2004; Lee, 2012; Morandi et al., 2019; Manik et al., 2022). We show that this bacterial toxin, designated ARC^tox, shares the catalytic residues, enzymatic activity, and overall fold of CD38, yet functions as a cytotoxic effector capable of depleting NAD^+ and NADP^+ in both bacteria and eukaryotic target cells, suggesting a dual role in microbial competition and host 88
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interaction. By tracing the distribution of ARC-like domains across bacterial genomes, we
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reveal that CD38-like folds are found in a wide range of bacteria and fused to diverse secretion
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and delivery systems, highlighting the remarkable evolutionary plasticity of PT.

Together, our findings indicate that CD38-like NAD⁺ hydrolase domains are not restricted to
eukaryotes, but also occur as components of bacterial polymorphic toxins, where they can be
used in bacterial warfare. This expands the known functional repertoire of PT and provide
insight into how similar enzymatic folds can be employed for distinct biological roles.
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105

106 **RESULTS**

107 **The C-terminal domain of the *Pantoea ananatis* PAAR PANA_2924 protein is toxic when**
108 **expressed in the *E. coli* or yeast cytosol**

109 The plant pathogen *Pantoea ananatis* strains LMG 2665T and DZ-12 use a type VI secretion
110 system (T6SS) to promote virulence in onion plants and to outcompete various bacterial
111 species, including *Escherichia coli* (Shyntum et al., 2015; Zhao et al., 2024). Other *Pantoea*
112 species encode up to three distinct T6SS gene clusters, each containing all essential structural
113 components, as well as a variety of *vgrG*, *hcp*, and *PAAR* islands (de Maayer et al., 2011;
114 Shyntum et al., 2014; Weller-Stuart et al., 2017). In addition to T6SS needle components, these
115 genomic islands often encode effector proteins, including the recently characterized TseG
116 nuclease (Zhao et al., 2024).

117 In the *P. ananatis* reference strain LMG 20103, one such *PAAR* island comprises three genes
118 encoding a putative EagR-family chaperone (PANA_2925), which usually binds to and protects
119 effector transmembrane domains (Alcoforado Diniz et al., 2015; Quentin et al., 2018; Ahmad
et al., 2020; Jurenas et al., 2021b), a polymorphic PAAR protein with an extended C-terminal region of unknown function (PANA_2924), and a small protein (PANA_2923) (Figure 1A). This gene organization resembles canonical chaperone-dependent polymorphic toxin operons, where a central toxin gene fused to a trafficking domain is flanked by a chaperone and an immunity protein.

Structural predictions using AlphaFold3 and BLASTp searches revealed that PANA_2924 is comprised of three distinct domains: a split PAAR domain (residues 1–21 (PAAR' or pre-PAAR) and 72–168 ('PAAR)), which could associate with the T6SS needle spike, a five-helix bundle (residues 22–71 and 169–249), and a globular C-terminal extension (PANA_2924-C, residues 255–387) (Figures 1B and 1C). It is noteworthy that the PAAR domain is interrupted by two helices from the central bundle (Figure 1B), yielding PAAR' (or prePAAR) and 'PAAR regions, as previously observed (Ahmad et al., 2020; Jurènas et al., 2021b).

To test whether the C-terminal extension corresponds to a toxin module, PANA_2924-C was cloned under the control of the *P*<sub>lac</sub> promoter into the low-copy pNDM220 vector, and its toxicity was tested in *E. coli*. While PANA_2924-C was not toxic when its expression was repressed by the presence of glucose, induction with IPTG led to strong growth inhibition, indicating antibacterial activity in the *E. coli* cytosol (Figure 1D). As the *P. ananatis* T6SS is involved in virulence towards onions (Shynthum et al., 2015; Zhao et al., 2024), we tested whether the PANA_2924-C was also toxic to eukaryotic cells. The same domain was expressed in *Saccharomyces cerevisiae* from the single-copy pRS416 plasmid. We were however unable to clone the PANA_2924-C into pRS416, likely due to its toxicity and plasmid leakiness in *E. coli* during the cloning procedure, but we obtained a clone (PANA_2924-C*, see Experimental Procedures) that is likely attenuated in its toxicity. Yeast growth was significantly inhibited upon induction of PANA_2924-C* expression (Figure 1E), demonstrating that the C-terminal
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extension of the PANA_2924 specialized PAAR protein has dual antibacterial and anti-eukaryotic activity.

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PANA_2923 encodes an immunity protein that neutralizes PANA_2924 toxicity through protein-protein interaction

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To determine whether the downstream gene, PANA_2923, encodes a cognate immunity protein conferring protection against PANA_2924, it was cloned under the *ParaBAD* promoter in the pBAD33 vector and co-expressed with the toxic PANA_2924 C-terminal domain. Co-expression fully rescued *E. coli* growth (Figure 1D), demonstrating that PANA_2923 neutralizes the toxin's activity.

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Co-purification assays using affinity chromatography confirmed a direct interaction between untagged PANA_2923 and 6×His-TEV-tagged PANA_2924 C-terminal domain (Figure 2A).

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This complex was stable enough to resist TEV cleavage and size exclusion chromatography (SEC) (Figure 2B). However, the complex could be separated upon addition of 8M urea in the affinity chromatography wash buffer, which allowed the purification of the PANA_2924-C domain alone (Figure 2B). The isolated domain and the complex are eluted from the Superdex 75 SEC column with a volume of 13.1 mL and 12.3 mL (Figure 2C). Based on the calibration curve (Supplemental Figure S1), these elution volumes are consistent with molecular weights of 19.2 and 27.3 kDa for PANA_2924-C alone (theoretical mass: 16.5 kDa) and PANA_2924-C/PANA_2923 (theoretical mass: 25 kDa), respectively, suggesting a 1:1 stoichiometry between the toxin and its immunity protein.

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PANA_2924 C-terminal domain structure reveals an ADP-ribosyl cyclase fold
The purified complex of the PANA_2924 C-terminal domain and its PANA_2923 immunity was subjected to crystallization trials without success. Crystallization of the PANA_2924 extension was, however, achieved after dissociating the immunity protein from the complex by adding urea to the affinity chromatography washing buffer, followed by refolding and SEC purification of the isolated toxin (Figure 2B). An X-ray diffraction dataset was collected, and the structure was solved by molecular replacement using the AlphaFold3 model, followed by refinement to a resolution of 1.6 Å (Figure 3A, Supplemental Table S1). PANA_2924 crystallized in the space group P2₁2₁2₁, with two molecules per asymmetric unit. These two molecules adopt similar conformations (Supplemental Figure S2). However, while the first chain could be modeled from residues 252 to 385, missing a flexible loop from 311 to 317, the second chain could be modeled from residues 250 to 385, missing a different flexible loop (amino-acids 345 to 349) (Supplemental Figure S2). The structure reveals a globular fold with two lobes, consisting of a four-stranded β-sheet covered by a helical cap composed of five α-helices (Figure 3A). A search for structural homologues using DALI (Holm, 2022) showed that the PANA_2924 C-terminal extension displays a fold similar to the human protein CD38 (Z-score: 8.6). Although the overall identity of the PANA_2924-C and CD38 domains retrieved using DALI is relatively low (26.3%) (Supplemental Figure S3A), the two domains are indeed structurally similar (Figure 3B). CD38 is a multifunctional protein: CD38 is a receptor at the surface of immune cells, responsible for the activation of T cells to produce cytokines (Piedra-Quintero et al., 2020). CD38 has also ADP ribosyl cyclase (ARC) activity: it hydrolyzes NAD⁺ to generate cyclic ADP ribose (cADPr), a messenger mediating various physiological functions in eukaryotic (notably involved in calcium signaling) and bacterial cells (Schuber and Lund, 2004; Lee, 2012; Ferrero et al., 2014; Takasawa, 2022; Lee et al., 2022). Importantly, key catalytic residues of the CD38 ARC domain – two glutamates and two tryptophans located in the cavity between the β-sheet and the α-helical cap (Munshi et al., 1999; Yamamoto-Katayama
et al., 2002; Schuber and Lund, 2004; Kuhn et al., 2014, Supplemental Figure S3B) – are conserved in the PANA_2924 ARC toxin (hereafter named ARC^tox^) (Figure 3C). These residues (W273, E299, W323, E356) form a putative active site. Indeed, substitution of any of the ARC^tox^ putative catalytic residues, W273, E299, W323 and E356, by alanines abolished ARC^tox^ activity in *E. coli* (Figure 3D). In agreement with this information, the AlphaFold3 model of the ARC^tox^ with its immunity protein PANA_2923 (ARC^imm^), predicted with high confidence (Supplemental Figure S4), suggests that ARC^imm^ interacts extensively with the ARC^tox^ catalytic cleft (interface area of 1130 Å), with a loop projecting into the active site (Figures 3E and 3F).

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202 ARC^tox^ hydrolyzes NAD⁺ and NADP⁺ *in vitro* and *in vivo* but does not produce cADPr

203 We next assessed the enzymatic activity of the purified, refolded, ARC^tox^ domain. *In vitro* assays showed efficient degradation of both NAD⁺ and NADP⁺ (Figure 4A). NAD⁺ degradation activity was confirmed *in vivo*, by measuring total NAD⁺ levels in *E. coli* cells after 1 hour of induction of the ARC^tox^ domain: Figure 4B shows that expression of ARC^tox^ led to a drastic decrease of cellular NAD⁺ levels, confirming enzymatic activity in the cytosol. Co-expression of ARC^imm^ or expression of the ARC^tox^ catalytically inactive mutants restored NAD⁺ levels to near baseline, except for the E299A mutant, which retained a level of degradation comparable to that of the wild-type toxin.

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211 To get further information on the reaction catalyzed by ARC^tox^, we performed high-pressure 212 liquid chromatography (HPLC) to identify the reaction products. Upon incubation with ARC^tox^, 213 NAD⁺ and NADP⁺ were converted into nicotinamide and ADP ribose (Figure 4C, left panels). 214 No cADPr was, however, detected, likely due to its immediate hydrolysis to ADP ribose (Sauve 215 et al., 1998). Consistent results were obtained with the well-characterized human CD38 ADP
216 ribosyl cyclase (Figure 4C, middle panels), further supporting functional conservation. These
217 products were specifically due to ARCtox activity, as (1) mutation of the ARCtox E356 catalytic
218 residue prevented NAD+ and NADP+ hydrolysis (Figure 4C, right panels), (2) NAD+ and
219 NADP+ did not spontaneously hydrolyze during the assay (Supplementary Figure S5A), and
220 (3) no nucleotide was detected by HPLC in the ARCtox protein preparations (Supplementary
221 Figure S5B).

222

223 ARC domains are widespread and associated with polymorphic toxin systems

224 To explore the evolutionary context of ARC^tox, we searched for homologous domains across
225 bacterial genomes. ARC-like domains were found throughout the bacterial tree of life (Figures
226 5A and 5B), exclusively associated with secretion-targeting domains such as PAAR, RHS, FIX
227 (T6SS), LXG and EsxA (T7SS), TPS (two-partner T5SS), and other extracellular or cell-wall
228 anchoring elements like pyocins, Flp pilins, and fibronectin type III (Fn3) domains (Schneider
229 et al., 2013; Whitney et al., 2017; Jana et al., 2019; Wood et al., 2019; Jurenas et al.,
230 2021b)(Figure 5A). Structural modeling showed that all these ARC homologs adopt the typical
231 CD38-like ADP ribosyl cyclase fold (Supplemental Figures S6 and S7).

232 Based on amino-acid sequence identity (>70%), the closest homologues of *P. ananatis* ARC^tox
233 are encoded within *Listeria* spp genomes and systematically associated with T7SS-dependent
234 LXG domains. Toxicity assays of a representative T7SS ARC domain from *Listeria*
235 *monocytogenes* (LisARC and ARC^tox amino-acid identity / similarity: 68.6 / 82.5 %) confirmed
236 that it is toxic when expressed in *E. coli* and was neutralized by its cognate immunity protein
237 (Figure 5C). Further cross-protection experiments demonstrated that both *Pantoea* ARC^tox and
238 LisARC are neutralized by non-cognate immunity proteins (Figure 5C, LisARCimm and
239 ARC^imm amino-acid identity / similarity: 38.2 / 67.1 %). The high identity between ARC^tox and
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LisARC, and the cross-neutralization by their immunities suggest that ARC domains have been
recently recruited to different secretion systems.

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ARC<sup>tox</sup> can be secreted via a heterologous delivery system

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To test whether ARC<sup>tox</sup> could be redirected through another delivery system, we engineered a chimera using the *E. coli* CdiAB contact-dependent inhibition (CDI) system, as done recently with the *Xenorhabdus bovieni* TreX ADP-ribosyl transferase (*XbART*, Dumont et al., 2024).

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For this, ARC<sup>tox</sup> was fused to the C-terminal CdiA stick domain, after the conserved VENN motif that delimitates CdiA transport and toxic domains (Figures 6A and 6B). Exposition of this chimeric effector to the *E. coli* DH5α cell surface was toxic to *E. coli* W3110 competitor cells but not to cells expressing the ARC immunity protein, demonstrating effective delivery and functional toxin activity (Figure 6C). In contrast, ARC<sup>imm</sup> did not protect against an unrelated CDI toxin (*XbART*), indicating specific poisoning of recipient cells with ARC<sup>tox</sup> (Figure 6C).

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DISCUSSION

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In this manuscript, we report the characterization of a novel and conserved class of versatile polymorphic toxin domains, ARC<sup>tox</sup>, sharing structural and functional characteristics of the human CD38 ADP ribosyl cyclase. We demonstrate that the C-terminal extension of a specialized PAAR protein from *Pantoea ananatis*, PANA_2924, acts as a potent cytotoxic effector, exhibiting both antibacterial and anti-eukaryotic activity through depletion of the cellular NAD<sup>+</sup>/NADP<sup>+</sup> pool. This toxin is neutralized by a tightly binding immunity protein

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263 (PANA_2923), forming a canonical toxin-immunity module reminiscent of those employed in
264 interbacterial conflict. Very recently, the ARC antibacterial Tac1 toxin associated with the
265 *Streptococcus parasanguinis* T7SS was functionally and structurally characterized,
266 demonstrating that it hydrolyzes NAD⁺ and NADP⁺ into ADP-ribose and ADP-ribose 2'-
267 phosphate, respectively (Colautti et al., 2025).

268 Remarkably, structural and biochemical data indicate that ARCtox and Tac1 are bacterial
269 analogs of human CD38, a multifunctional enzyme involved in immune signaling and NAD
270 metabolism (Lee et al., 2022; Takasawa, 2022; Colautti et al., 2025), revealing an unexpected
271 link between polymorphic bacterial effectors and eukaryotic cell biology. The discovery of this
272 relationship between ARCtox and Tac1, and CD38 is significant both structurally and
273 functionally. The ARC toxins adopt a globular fold closely resembling CD38, characterized by
274 a β-sheet core capped by α-helices. DALI analysis and high-resolution crystal structures reveal
275 that these enzymes share not only overall architecture but also precise placement of key
276 catalytic residues – two glutamates and two tryptophans – which define the active site of the
277 CD38 family (Munshi et al., 1999; Schuber and Lund, 2004; Kuhn et al., 2014). Mutational
278 inactivation of these residues in ARCtox completely abrogates its toxic activity, confirming their
279 critical role in catalysis. Importantly, this enzymatic function is not merely structural mimicry:
280 ARCtox directly hydrolyzes NAD⁺ and NADP⁺ into ADP-ribose and nicotinamide,
281 recapitulating the enzymatic outcome of CD38 activity in eukaryotic cells (Schuber and Lund,
282 2004). The absence of cyclic ADP-ribose (cADPr), likely due to its rapid hydrolysis, aligns
283 with the catalytic profile of some CD38-like enzymes that favor hydrolysis over cyclization
284 (Sauve et al., 1998). These data also suggest that ARCtox antibacterial toxicity is not due to the
285 accumulation of cADPr, which could impact undefined signaling pathways in bacteria, but
286 rather poisons target cells by depleting the pool of NAD⁺/NADP⁺. Indeed, the recent work of
287 Colautti et al. demonstrated that the *S. parasanguinis* T7SS Tac1 ARC toxin hydrolyzes NAD⁺
with a rate of 12,000 molecules/min (Colautti et al., 2025), which is comparable with previous NAD glycohydrolase toxins (Whitney et al., 2015; Tang et al., 2018). The ability of a bacterial toxin to deplete NAD⁺, a central metabolic cofactor, highlights a sophisticated mechanism of intercellular antagonism that targets core bioenergetic processes. While many known bacterial toxins act by degrading nucleic acids, lipids or proteins (Zhang et al., 2012; Russell et al., 2014; Durand et al., 2014; Jurenas and Journet, 2021), ARC<sup>tox</sup> represents a distinct class of metabolic toxins, sabotaging host or competitor metabolism through NAD⁺ depletion. Other toxins belonging to the ART-fold superfamily have been shown to utilize the NAD⁺ or NADP⁺ for ADP-ribosylation of protein or of RNA targets, or for NAD⁺ hydrolysis (Suarez et al., 2010; Whitney et al., 2015; Palazzo et al., 2017; Ting et al., 2018; Tang et al., 2018; Jurenas et al., 2021a; Roussin and Salcedo, 2021; Jurenas et al., 2022; Bullen et al., 2022). This ART-fold superfamily comprises a conserved core fold corresponding to a central split β-sheet with a conserved order of strands (Aravind et al., 2015). The ARC fold is highly distinct, but our genomic analysis showed that it is also highly widespread, but previously only reported in eukaryotic CD38. In any case, the NAD⁺ depletion strategy likely confers a competitive advantage in polymicrobial environments, especially in the plant rhizosphere or during host colonization, where nutrient access and niche competition are intense (Hibbing et al., 2010; Ghoul and Mitri, 2016; Venturi and Keel, 2016). The dual activity of ARC<sup>tox</sup> against both bacterial and eukaryotic cells further suggests that this effector may function during both interbacterial competition and plant-pathogen interactions, consistent with the known virulence roles of *P. ananatis* T6SS in onion infection (Shyntum et al., 2015; Zhao et al., 2024).

The conservation between ARC<sup>tox</sup> and CD38 extends beyond structure and catalysis; it raises fascinating evolutionary questions. The phylogenetic distribution of ARC domains across diverse bacterial lineages, and their consistent association with modular toxin architectures (PAAR, RHS, LXG, TPS, Fn3, etc.), suggests that these domains have been evolutionarily co-
313 opted for secretion via multiple delivery systems, including T6SS, T5SS, T7SS, and CDI, or
314 for cell surface exposition. This modularity, which is a hallmark of polymorphic toxins, enables
315 effector domains to be flexibly redeployed across different antagonistic contexts. Yet the fact
316 that the same enzymatic fold underlies both a eukaryotic signaling enzyme and a prokaryotic
317 toxin raises the possibility of an ancient common ancestry or repeated events of horizontal gene
318 transfer followed by domain repurposing. An additional possibility is that the similarity
319 between ARCtox and CD38 arose through convergent evolution, with both proteins
320 independently evolving similar fold and catalytic mechanism due to functional constraints on
321 NAD+ metabolism. The metabolic versatility of the CD38/ARC fold, capable of catalyzing both
322 cyclization and hydrolysis reactions on NAD+ substrates, may have facilitated its functional
323 diversification across kingdoms.

324 Our findings also highlight the high adaptability of bacterial effector systems. The
325 incorporation of ARCtox into a heterologous CDI system, where it successfully mediates
326 contact-dependent killing of target cells, highlights the modularity and portability of these
327 effectors. This property, combined with the cross-reactivity of immunity proteins (i.e., cross-
328 protection between *Pantoea* and *Listeria* ARCtox modules), suggests that ARCtox domains can
329 be rationally engineered for synthetic biology applications, such as programmable antibacterial
330 agents, targeted delivery systems, or novel biocontrol strategies in agriculture. Moreover, the
331 cross-protection between non-cognate immunity proteins may reflect conserved structural
332 features of the catalytic cleft, hinting at convergent evolution or selective pressure to maintain
333 compatibility within toxin-immunity networks.

334 The biological implications of ARCtox extend to host-pathogen interactions as well. Given its
335 demonstrated anti-eukaryotic activity in yeast, one may hypothesize that ARCtox-like proteins
336 are deployed during plant infection, where they could modulate host immunity, disrupt cellular
337 metabolism, or interfere with NAD+-dependent signaling pathways. Such roles would mirror
338 the function of CD38 in immune cells, albeit in a pathogenic rather than physiological context.

339 Further investigations into the role of ARCtox in plant-microbe interactions, including its
340 potential delivery into plant cells or modulation of host NAD+ pools, could reveal novel
341 mechanisms of bacterial virulence.

342 Our broader genomic survey of ARC domain distribution indicates that this ARC NAD
343 hydrolase toxin architecture is widespread across the bacterial tree of life. Its association with
344 diverse secretion and anchoring domains supports the notion that ARCtox is a core component
345 of the bacterial competitive arsenal. The presence of ARC domains in systems as distinct as
346 pyocins and T7SS suggests that ARCtox may contribute to antagonism in a variety of ecological
347 contexts, from soil and plant microbiomes to animal hosts and clinical infections.

348

349 EXPERIMENTAL PROCEDURES

350 Strains, media and growth conditions

351 *Escherichia coli* strain DH5α was used for cloning, toxicity tests, and NAD/NADH-Glo™ Assay (Promega). *E. coli* BL21 (DE3) and W3110 strains were used for protein production and bacterial competition, respectively. *E. coli* cells were grown at 37°C in Lysogeny Broth (LB) with agitation or on LB agar (1.5%) plates. When needed, media were supplemented with ampicillin (50-100 µg.mL⁻¹), chloramphenicol (30 µg.mL⁻¹), kanamycin (50 µ g.mL⁻¹), or streptomycin (100 µ g.mL⁻¹). Gene expression was induced by the addition of 1% of L-arabinose, 50-100 µM of IPTG or repressed with 1% of glucose. *Saccharomyces cerevisiae* strain W303 was used for toxicity assays. *S. cerevisiae* cells were grown at 30°C in YPAD (Yeast extract Peptone Adenine Dextrose) or SD (Synthetic Dextrose minimal) media with agitation, or on YPAD, SD, or SG (Synthetic Galactose minimal) agar plates.

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362 Plasmid construction and mutagenesis

363 All cloning procedures were performed by restriction-ligation or restriction-free based
364 methods. Oligonucleotides were purchased from IDT. DNA fragments encoding ARCtox and
365 ARCimm were amplified from *Pantoea ananatis* LMG 20103 using the Q5 polymerase (NEB).
366 Vector backbones and full plasmid templates were amplified using PrimeSTAR Max DNA
367 polymerase (Takara). DNA fragment encoding the *Listeria monocytogenes* LS1292 LXG ARC
368 domain (LisARC) and its cognate immunity (LisARCimm) was synthesized by Twist
369 Bioscience. LisARC and LisARCimm fragments were then PCR-amplified using the Q5
370 polymerase. PCR products were purified using NucleoSpin Gel and PCR Clean-up columns
371 (Macherey-Nagel), digested with the appropriate restriction enzymes (NEB), and repurified
372 prior to ligation. Ligated plasmids were transformed into *E. coli* DH5α and screened by colony
373 PCR using EconoTaq PLUS GREEN 2x Master Mix (Biosearch Technologies). Plasmid DNA
374 was extracted using either the Wizard Plus SV Minipreps kit (Promega) or the NucleoSpin
375 Plasmid kit (Macherey-Nagel) and verified by Sanger sequencing (Eurofins). Constructs were
376 selected on LB agar plates containing ampicillin (for pRS416_Gal1, pNDM220, and pET-
377 Duet1), chloramphenicol (for pBAD33), kanamycin (for pRSF-Duet1), or streptomycin (for
378 pCDF-Duet1). Glucose (1%) was added for selection with the pNDM220 vector. Note that,
379 despite several attempts, we never succeeded to clone the wild-type sequence of PANA_2924
380 C-terminal domain into pRS416_Gal1, probably due to its high toxicity and promoter leakiness
381 in *E. coli*. However, we obtained an attenuated mutant bearing a 1 bp (A) deletion at position
382 435. Toxin and immunity operon were cloned into pET-Duet1, while immunity genes were also
383 cloned individually into pBAD33, pRSF-Duet1, or pCDF-Duet1. Site-directed mutagenesis of
384 ARCtox catalytic residues was performed using QuickChange mutagenesis by PCR-amplifying
385 the whole plasmid using primers bearing the desired mutations and the pNDM220-ARCtox
386 plasmid as template. PCR products were cleaned up from gels to eliminate the template
387

plasmid, then phosphorylated and ligated (T4 DNA ligase, NEB) prior to transformation. The
pCH10163-ARC^tox-ARC^imm plasmid, encoding ARC^tox fused to *E. coli* CdiA was generated by
restriction-free cloning, using overlapping regions to fuse the desired fragments.

388

389

390

391

## Toxicity assays in *E. coli* and yeast

392

*E. coli* DH5α cells were co-transformed with the pNDM220 and pBAD33 plasmid pairs bearing either ARC^tox, ARC^tox mutants or LisARC and ARC^imm or LisARC^imm, respectively, or empty vectors as controls. Transformants were selected on LB agar plates supplemented with ampicillin, chloramphenicol, and 1 % of glucose. Overnight cultures grown in the presence of antibiotics and glucose were diluted in 10-fold series, and 10-µL drops were spotted on LB agar plates containing antibiotics and either 1 % glucose (repression conditions) or 1 % arabinose (immunity induction from pBAD33) with 0.05 mM or 0.02 mM IPTG (toxin induction from pNDM220). Plates were incubated at 37 °C for 16-20 hours before imaging.

393

394

395

396

397

398

399

400

*S. cerevisiae* strain W303 was transformed with the pRS416_Gal1 encoding ARC^tox or an empty vector as control and selected on SD agar plates. Overnight cultures grown in SD liquid medium were diluted in 10-fold series, and 5-µL drops were spotted onto SD (repression condition) and SG (induction condition) agar plates. Plates were incubated at 30 °C for 44-48 hours before imaging.

401

402

403

404

405

406

## Purification of ARC^tox-ARC^imm complex and ARC^tox

407

Freshly transformed *E. coli* BL21 (DE3) cells carrying pET-hisTEV-ARC^tox-ARC^imm, pRSF-ARC^imm, and pCDF-ARC^imm or pET-hisTEV-ARC^tox-E356A were grown overnight in LB medium supplemented with appropriate antibiotics. Cultures were diluted 1:100 into 2 L of LB and grown at 37°C to an OD₆₀₀ of 0.8. After addition of 0.5 mM of IPTG, cells were grown overnight at 16°C with agitation. Cells were collected by centrifugation at 4,000 × g and

408

409

410

411
412 resuspended in lysis buffer (50 mM Tris-HCl pH 8.5, 250 mM NaCl, 1 mM TCEP) containing
413 cOmplete™ protease inhibitor cocktail (Sigma-Aldrich). Cells were lysed by sonication, and
414 the cell lysate was clarified by centrifugation at 20,000 × g for 45 min. The supernatant was
415 filtered through a 0.45 µm filter and incubated with 2 mL TALON Metal Affinity Resin
416 (Takara) pre-equilibrated with lysis buffer for 1 h at 4 °C with gentle mixing. The resin was
417 washed five times with 15 bed volumes of lysis buffer, and bound proteins were eluted with
418 1 mL of elution buffer (50 mM Tris-HCl pH 8.5, 250 mM NaCl, 1 mM TCEP, 200 mM
419 imidazole). For purification of ARC toxin alone, after the washing steps with lysis buffer, the
420 resin was incubated with 8 M urea for 2 min, followed by one additional wash with 8 M urea.
421 Refolding was performed by sequential washes with refolding buffer 1 (50 mM Tris-HCl, pH
422 8.5, 125 mM NaCl, 5% glycerol), followed by two washes with refolding buffer 2 (50 mM Tris-
423 HCl pH 8.5, 250 mM NaCl, 1% glycerol). The toxin was then eluted using 1 mL of elution
424 buffer. The His-TEV tag was removed from both the ARC^tox/ARC^imm complex and ARC^tox by
425 incubation with the purified TEV protease (1:100 molar ratio of TEV protease:His-tagged
426 protein) overnight at 4 °C. Proteins were further purified by size-exclusion chromatography
427 (SEC) using a Superdex 75 Increase 10/300 GL column (Cytiva) equilibrated in buffer A
428 (20 mM Tris-HCl pH 8.5, 150 mM NaCl, 1 mM TCEP) for the ARC^tox/ARC^imm complex, or
429 buffer B (20 mM Tris-HCl pH 8.5, 150 mM NaCl) for ARC^tox. The SEC column was calibrated
430 using Gel filtration Standard (Biorad), which includes bovine thyroglobulin (670 kDa), bovine
431 γ-globulin (158 kDa), chicken ovalbumin (44 kDa), horse myoglobin (17 kDa), and vitamin
432 B₁₂ (1.35 kDa) (Supplemental Figure S1).

433

434 **Crystallization, structure determination and refinement of ARC**<sup>tox</sup>

435 Following purification, tag removal, and gel filtration, ARC^tox toxin was concentrated to
436 13.4 mg.mL⁻¹ and subjected to crystallization screening using the sitting-drop vapor diffusion
method at 20°C. Crystallization drops were set up in Swissci 96-well 2-drop MRC plates (Molecular Dimensions) by mixing 0.5 µL of protein solution with 0.5 µL of reservoir solution. Drops were equilibrated against 70 µL of crystallization screens, including Crystal Screen I and II (Hampton Research), LMB, PACT Premier, JCSG+, and MemGold (Molecular Dimensions). Diffraction-quality crystals of ARCtox were obtained using a reservoir solution containing 0.2 M ammonium chloride, 0.1 M sodium acetate (pH 5.0), and 20% (w/v) PEG 6000. Prior to cryo-cooling in a liquid nitrogen stream, crystals were cryo-protected by briefly transferring them into reservoir solution supplemented with 20% glycerol. X-ray diffraction data were collected at the PROXIMA-2A (PX2A) beamline at the SOLEIL Synchrotron (Gif-sur-Yvette, France), integrated with XDS (Kabsh, 2010) and scaled with AIMLESS (Evans et al., 2013) from the CCP4 program suite (Hough et al., 2018). The structure was solved by molecular replacement using a AlphaFold3 model of ARCtox as a search probe using PHASER (McCoy et al., 2007) in PHENIX (Adams et al., 2010), with two molecules in the asymmetric unit. The initial map was excellent, and the model was manually improved in COOT (Casanal et al., 2020) and subsequently refined with PHENIX (Adams et al., 2010). The final model was refined to a Rwork/Rfree of 0.18/0.21 with excellent statistics (Supplemental Table S1). The coordinates and structure factors were deposited in the Protein Data Bank (PDB) with accession code 9RO8.

454

455

### *In vitro* NAD(P)⁺ hydrolysis assay

Reactions were performed in 100 µL of PBS supplemented with 0.6 mM of NAD⁺ or of NADP⁺ (Roche). Purified, refolded ARCtox was added to a final concentration of 0.1 µM. Samples were incubated at room temperature for 30 min. The reaction was then quenched by adding 50 µL of 6 M NaOH. Fluorescence was measured using TECAN plate reader (excitation: 340 nm; emission: 420 nm) after 30 min of incubation at room temperature in the dark.

461
462 **In vivo NAD/NADH-Glo assay**

463 *E. coli* strain DH5α cells producing wild-type and mutant ARC^tox^ domains, or both ARC^tox^ and
464 ARC^imm^ were grown in medium supplemented with antibiotics and 1% glucose at 37°C with
465 agitation until OD₆₀₀ reached 0.5. Cells were then washed with LB medium and resuspended in
466 fresh LB supplemented with 0.05 mM of IPTG, 1 % of arabinose, and antibiotics. After
467 incubation for 1 hour at 37°C with agitation, 10⁹ cells were mixed with the NAD/NADH-Glo™
468 detection reagent (Promega), and the measure of NAD⁺ levels was done according to the
469 manufacturer's instructions.

470

471 **Analysis of ARC<sup>tox</sup> activity by High Pressure Liquid Chromatography**

472 NAD⁺ or NADP⁺ (1.5 mM) were incubated for 30 min with 0.1 µM of wild-type or E356A
473 variant ARC<sup>tox</sup> or of purified CD38 soluble domain (kind gift of Alain Roussel, LISM,
474 Marseille, France), or with buffer alone. As further control, 0.1 µM of ARC<sup>tox</sup> was incubated
475 with buffer alone to ensure no contamination of ARC<sup>tox</sup> preparations with nucleotides. Proteins
476 were then retained onto Amicon Ultra 3 kDa filters (Sigma-Aldrich) by centrifugation at 11,000
477 × g for 15 min. The flow-through was injected into an Agilent 1260 Infinity HPLC system
478 equipped with a mixed-mode RP/anion-exchange Atlantis PREMIER BEH C₁₈ AX column.
479 Analytes were separated at a flow rate of 1 mL.min⁻¹ for 40 min, with a gradient profile of 100%
480 Solvant A (10 mM ammonium formate pH3.0 for NADP⁺, 20 mM ammonium formate pH2.9
481 for NAD⁺) for 5 min, linear gradient to 50% Solvant A / 50% Solvent B (acetonitrile) for 30
482 min, and 100% Solvent B for 5 min. Analytes were detected by absorbance at 254 nm. The
483 retention times were compared to standards run on the same column (Nicotinamide (NAM)
484 250-450 µM, NAD⁺ 40 µM, NADP⁺ 150 µM, ADP-ribose 150-200 µM, cyclic ADP-ribose 60-
485 160 µM)

486
487 Bacterial competition assay

488 Interbacterial competition was measured using the LAGA assay (Taillefer et al., 2023). This
489 assay is based on the degradation of chlorophenol-red β-D-galactopyranoside (CPRG), a
490 membrane-impermeable chromogenic substrate of the β-galactosidase, which is cleaved by the
491 β-galactosidase released by the recipient cell lysis. *E. coli* DH5α cells producing CdiB and the
492 CdiA chimera proteins were used as donors whereas *E. coli* W3110 (*lacZ*⁺) cells were used as
493 recipient. Both strains were grown separately to an OD₆₀₀ of 0.5, washed twice with LB medium
494 supplemented with 0.05 mM IPTG, and mixed to a 1:2 (donor:recipient) ratio. Ten µL of the
495 mixtures were spotted onto LB agar plates supplemented with 0.05 mM of IPTG. Plates were
496 incubated for 4 hours at 37 °C, and recipient cell lysis was observed by the addition of a 10-µL
497 drop of 2 mM of CPRG (Roche) on the top of each spot.

498

499 Statistical analysis

500 *In vitro* ARC⁹ₓ activity data were analyzed using an unpaired *t*-test. *In vivo* data from
501 NAD/NADH-Glo™ assay were analyzed using one-way ANOVA followed by Dunnett's post
502 hoc test. All analyses were performed using the GraphPad Prism software.

503

504 DATA AVAILABILITY

505 The *Pantoea ananatis* PANA_2924 ARC⁹ₓ structure has been deposited to the Protein Data
506 Bank (PDB) under accession code 9RO8.

507

508 SUPPORTING INFORMATION
509 This article contains supporting information (seven Supplemental Figures and four

510 Supplemental Tables).

511

512 ACKNOWLEDGMENTS

513 We thank the members of the Cascales and Jurènas laboratories for discussions and support,

514 Vincent Géli, Pierre Luciano and Frédéric Jourquin (CRCM, Marseille, France) for providing

515 yeast strains, vectors and protocols, Alain Roussel (LISM, Marseille, France) for the kind gift

516 of purified human CD38, the staff at the SOLEIL PROXIMA 2 beamline, and Audrey Gozzi

517 for technical support.

518

519 FUNDING INFORMATION

520 This work was funded by the Centre National de la Recherche Scientifique, the Aix-Marseille

521 Université, and by grants from the Agence Nationale de la Recherche (ANR-20-CE11-0017),

522 and the Fondation Bettencourt Schueller to EC. JM's work was supported by the ANR-20-

523 CE11-0017 grant.

524

525 CONFLICT OF INTEREST

526 The authors declare no competing interests.

527

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715 systems: Comprehensive characterization of trafficking modes, processing, mechanisms of
716 action, immunity and ecology using comparative genomics. *Biol Direct.* **7**, 18.
717 Zhao X, Gao L, Ali Q, Yu C, Yuan B, Huang H, Long J, Gu Q, Wu H, Gao X. (2024) A type
718 VI secretion system effector TseG of *Pantoea ananatis* is involved in virulence and
719 antibacterial activity. *Mol Plant Pathol.* **25**, e13442.

720
721 LEGENDS TO FIGURES

722 **Figure 1.** *Pantoea ananatis* PANA_2924 C-terminal domain is toxic in *E. coli* and yeast
cells. (A) Schematic representation of the *P. ananatis* PANA_2925-PANA_2923 PAAR island.
723
724 Genes encoding the EagR chaperone (PANA_2925), the specialized PAAR protein
725 (PANA_2924), and a protein of unknown function (PANA_2923) are depicted in white, light
726 flesh and pink, respectively. (B and C) Schematic representation (B) and AlphaFold3 structural
727 model (C) of the PANA_2924 specialized PAAR protein, highlighting the different domains
728 and their boundaries: PAAR domain (PAAR' or prePAAR in light red, and 'PAAR in yellow),
729 trans-membrane domain (TMD' and 'TMD in light green and green, respectively) and C-
730 terminal extension (blue). (D) Toxicity assay in the heterologous host *E. coli*. Overnight
731 cultures of *E. coli* cells expressing the PANA_2924 C-terminal extension (2924-C) from the
732 low-copy vector pNDM220, PANA_2923 (2923) from the pBAD33 vector, or both (2924-
733 C+2923) were serially diluted (10⁻¹ to 10⁻⁶) and spotted on LB agar plates, supplemented with
734 1% of glucose (left panel, 2924-C repression), with 0.05 mM of IPTG (middle panel, 2924-C
735 induction), or with 0.05 mM of IPTG and 1% of L-arabinose (right panel, 2924-C and 2923
736 induction). (E) Toxicity assay in yeast. Overnight cultures of W303 cells expressing the
737 attenuated PANA_2924-C* mutant from the pRS416_Gal1 vector were serially diluted and
738 spotted on SD (left panel, 2924-C repression) or SG (right panel, 2924-C induction) medium.
739

739

740 **Figure 2.** PANA_2924 C-terminal extension forms a complex with PANA_2923. (A) Pull-down assay. Soluble extracts of *E. coli* BL21(DE3) cells producing 6×His-TEV-2924-C and untagged PANA_2923, or untagged PANA_2923, were subjected to pull-down by immobilized metal affinity chromatography (IMAC). The cell extract (CE) and eluted material (E) were separated by 15%-acrylamide SDS-PAGE and stained with Coomassie blue. The positions of
the proteins are indicated on the right. Molecular weight markers (in kDa) are indicated on the left. (B) Purification of 2924-C/2923 complex and 2924-C alone. Soluble extracts of *E. coli* BL21(DE3) cells producing 6×His-TEV-2924-C and untagged PANA_2923 were subjected to IMAC purification, and gel filtration (SEC) after cleavage by the TEV protease. For 2924-C alone, the complex immobilized by IMAC was washed with 8M urea to dissociate and discard untagged 2923, prior to elution, TEV cleavage, and SEC. The fractions after IMAC, TEV cleavage and SEC were separated by 15%-acrylamide SDS-PAGE and stained with Coomassie blue. The positions of the proteins are indicated on the right. Molecular weight markers (in kDa) are indicated on the left. (C) Gel filtration analysis. The PANA_2924-C / PANA_2923 complex (green line) and the PANA_2924-C toxin alone (blue line) obtained after affinity purification and TEV cleavage were subjected to size-exclusion chromatography on a Superdex 75 Increase 10/300 GL column. The elution profile of molecular weight standards is shown as dotted line and the molecular weight of each of the standard is indicated on top (in kDa). Below the graph are shown the 15%-acrylamide SDS-PAGE and Coomassie blue staining analyses of selected SEC fractions encompassing the 2924-C/2023 (green, upper panel) and 2924-C (blue, lower panel) peaks. The calibration curve, linear regression, R² value and experimental molecular weights of the complex and toxin alone are shown in Supplemental Figure S1.

762

Figure 3. PANA_2924 C-terminal extension displays an ADP ribosyl cyclase (ARC) fold comparable to human CD38, with a typical catalytic cleft blocked by the PANA_2923 immunity protein. (A) Ribbon representation of the 1.6-Å resolution crystal structure of PANA_2924 C-terminal extension. α-helices are depicted in light blue whereas β-strands are shown in cyan. The surface structure is shown in grey transparent. N, N-terminus; C, C-terminus. (B) Structural comparison between *P. ananatis* PANA_2924 C-terminal extension (this work) and human CD38 protein (PDB: 2PGJ) in the same orientation. Conserved
secondary structures are colored as in panel A. N, N-terminus; C, C-terminus. The PANA_2924-C structure is shown rotated 180° relative to the vertical plane compared with panel A. The amino-acid alignment of PANA_2924-C and human CD38 is shown in Supplemental Figure S3A. (C) Close-up view of the PANA_2924-C putative active site, showing potential key residues as grey sticks. A comparison of PANA_2924-C and CD38 NAD⁺-binding pockets, including catalytic residues, is shown in Supplemental Figure S3B. (D) Toxicity assay in the heterologous host *E. coli*. Overnight cultures of *E. coli* cells expressing the wild-type (WT) or mutated ARC¹⁻⁰⁻⁸ from the low-copy vector pNDM220 were serially diluted (10⁻¹ to 10⁻⁶) and spotted on LB agar plates, supplemented with 1% of glucose (left panel, ARC¹⁻⁰⁻⁸ repression), or 0.05 mM of IPTG (right panel, ARC¹⁻⁰⁻⁸ induction). (E) Ribbon representations of the AlphaFold3 prediction model of the ARC¹⁻⁰⁻⁸/ARC⁴²⁻⁴⁵ complex (ipTM score = 0.92). The toxin domain is shown in blue, whereas the immunity is shown in pink, within the complex's surface representation colored based on residue electrostatic properties (from blue (positive) to red (negative)). pLDDT and PAE confidence scores of the ARC¹⁻⁰⁻⁸/ARC⁴²⁻⁴⁵ complex are presented in Supplemental Figure S4. (F) Contact site between ARC¹⁻⁰⁻⁸ and ARC⁴²⁻⁴⁵ highlighting the ARC⁴²⁻⁴⁵ loop penetrating into the ARC¹⁻⁰⁻⁸ catalytic cleft. The side chains of the ARC¹⁻⁰⁻⁸ catalytic residues are shown in grey. Residues within 4 Å of the ARC¹⁻⁰⁻⁸ and ARC⁴²⁻⁴⁵ interface are shown as sticks, and dotted grey lines indicate potential polar contacts.

**Figure 4.** *P. ananatis* ARC¹⁻⁰⁻⁸ depletes NAD⁺ and NADP⁺ pools *in vivo* and *in vitro*. (A) Fluorescence-based assay showing NAD⁺ and NADP⁺ levels in untreated (0) and ARC¹⁻⁰⁻⁸-treated samples. NAD⁺ (left panel) or NADP⁺ (right panel) (0.6 mM) were incubated with the buffer (0) or with refolded ARC¹⁻⁰⁻⁸ (0.1 µM) *in vitro* for 30 min. RFU – relative fluorescence units. Boxes indicate standard deviation of the mean of three replicates. **** denotes a
statistically significant difference between the two conditions, based on an unpaired t-test
(P<0.0001). (B) Luminescence assay measuring NAD+/NADH levels in E. coli DH5α cells
producing the wild-type (WT) or the indicated mutated ARCtox domains from the pNDM220
vector and ARCimm from the pBAD33 vector. RLU – relative luminescence units. Bars show
means ± SD from six independent experiments (same-color points represent replicates from the
same experiment). *** denotes statistically significant difference compared to the control group
(one-way ANOVA, post-hoc Dunnett's test, P<0.001). ns = not significant. (C) HPLC
chromatograms of the products of the reactions of NAD+ (upper panels) and NADP+ (lower
panels) in the presence of ARCtox (left panels), CD38 (middle panels) or of the ARCtox E356A
variant (right panels) subjected to mixed-mode RP/anion-exchange Atlantis Premier BEH C18
AX column. The products are shown with the black lines. Chromatograms of selected standard
analytes are shown in color (green, nicotinamide; orange, NAD+/NADP+; flesh, ADP ribose;
blue, cyclic ADP ribose). HPLC analysis controls used to assess NAD+/NADP+ spontaneous
hydrolysis and nucleotide contamination of ARCtox preparations are presented in Supplemental
Figure S5.

810

**Figure 5.** Active ARC toxin domains are widespread across bacteria. (A) Domain architectures of selected proteins containing ARC domain homologs (1, *Pantoea ananatis* WP_013026797; 2, *Agrobacterium rubi* NTF28035; 3, *Burkholderia gladioli* WP_186165364; 4, *Listeria monocytogenes* WP_120135998; 5, *Gordonia jinhuaensis* WP_188589035; 6, *Lactiplantibacillus plantarum* WP_076633504; 7, *Stenotrophomonas maltophilia* OCK46403; 8, *Nonomuraea fuscirosea* WP_364663299; 9, *Marinobacter shengliensis* WP_138437386; 10, *Pendulispora albinea* WP_394825747; 11, *Allomuricauda* sp. RPG31737; 12, *Streptomyces mirabilis* WP_388533272). ARC domains are indicated in bright colors. Comparisons of the AlphaFold3 structural models of each of these ARC domains with *P. ananatis* ARCtox, and their
statistics are shown in Supplemental Figure S6 and S7, respectively. (B) Cladogram of 1075 homologs of *P. ananatis* ARC^tox^. The positions of the selected ARC domains shown in panel A are indicated by the same color code. (C) Toxicity assay in the heterologous host *E. coli*. Overnight cultures of *E. coli* cells expressing the indicated ARC domain (*P. ananatis* ARC^tox^ or *L. monocytogenes* ARC, LisARC) from the low-copy vector pNDM220, and the indicated immunity proteins (*P. ananatis* ARC^imm^ and *L. monocytogenes* ARC^imm^) from the pBAD33 vector were serially diluted (10^0 to 10^6) and spotted on LB agar plates, supplemented with 1% of glucose (left panel, toxin repression), 0.02 mM of IPTG (middle panel, toxin induction), and 0.02 mM of IPTG and 1% of arabinose (right panel, toxin and immunity induction).

829

**Figure 6. CDI can be engineered to deliver ARC. (A) Schematic model of the mechanism of action of Contact-Dependent growth Inhibition (CDI) systems. The outer membrane component CdiB (light green) translocates CdiA (dark blue) to the donor cell surface. The receptor-binding domain of CdiA recognizes and binds to a specific receptor (grey) on the surface of the recipient cell. Then the translocon domain (flesh) delivers the toxin (blue) into the recipient cell. (B) Schematic representation of the CDI chimera engineered for ARC^tox^ delivery by the *E. coli* CdiAB CDI system. The cdiB and cdiA genes are shown in light green and dark blue, respectively. The grafted translocation domain and *P. ananatis* ARC^tox^ domain are shown in flesh and blue, respectively. ARC^imm^ is shown in pink. (C) Interbacterial competition assay. Donor (*E. coli* DH5α) and recipient (*E. coli* W3110) cells producing the indicated chimera CDIs (*P. ananatis* ARC^tox^ and *Xenorhabdus bovieni* ART) or ARC^imm^ were mixed at a 1:2 (v:v) ratio, spotted onto LB-agar, and incubated for 4 h at 37°C. Recipient cell lysis after competition was revealed using CPRG (yellow), which turns purple upon hydrolysis by β-galactosidase from lysed lacZ+ recipient cells. The killing efficiency heatmap (from yellow (no killing) to purple (killing)) is shown on the right.
<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></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" /></figure>

<figure><img src="image_8.png" /></figure>
<figure><img src="image_9.png" /><figcaption>A</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>B</figcaption></figure>

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

2924-C (PDB: 9RO8)
ARC<sup>tox</sup>

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

CD38 (PDB: 2PGJ)

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

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>
<figure><img src="image_18.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_19.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_20.png" /></figure>
A B

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>

C

empty
ARC^tox
ARC^tox / ARC^imm
LisARC
LisARC / LisARC^imm
ARC^tox / LisARC^imm
LisARC / ARC^imm

<figure><img src="image_23.png" /></figure>

glucose
(Tox repression)

<figure><img src="image_24.png" /></figure>

IPTG
(Tox induction)

<figure><img src="image_25.png" /></figure>

IPTG + ara
(Tox and Imm induction)
A

recipient cell

<figure><img src="image_26.png" /></figure>

<figure><img src="image_27.png" /></figure>

B

<figure><img src="image_28.png" /></figure>

C

donor cells (DH5α)

empty TreX ARC^tox ARC^tox TreX

<figure><img src="image_29.png" /></figure>

recipient cells (W3110)

<figure><img src="image_30.png" /></figure>
<table><caption>Supplemental Table 1. Data collection and refinement statistics.</caption><thead><tr><th></th><th>ARC<sup>tox</sup> (PDB : 9RO8)</th></tr></thead><tbody><tr><td><strong>Data collection</strong></td><td></td></tr><tr><td>Resolution range</td><td>39.66 - 1.594 (1.63 - 1.59)</td></tr><tr><td>Space group</td><td>P 21 21 21</td></tr><tr><td>Unit cell</td><td>44.23 70.213 96.119 90 90 90</td></tr><tr><td><strong>Total reflections</strong></td><td></td></tr><tr><td>Unique reflections</td><td>40577 (2746)</td></tr><tr><td><strong>Multiplicity</strong></td><td></td></tr><tr><td>Completeness (%)</td><td>99.73 (96.38)</td></tr><tr><td>Mean I/sigma(I)</td><td>17.0 (2.2)</td></tr><tr><td>Wilson B-factor</td><td>21.48</td></tr><tr><td>R-merge</td><td>0.092 (1.053)</td></tr><tr><td>R-meas</td><td>0.096 (1.099)</td></tr><tr><td>R-pim</td><td>0.027 (0.31)</td></tr><tr><td>CC1/2</td><td>0.99 (0.76)</td></tr><tr><td><strong>Refinement</strong></td><td></td></tr><tr><td>Reflections used in refinement</td><td>40577 (2746)</td></tr><tr><td>Reflections used for R-free</td><td>2000 (136)</td></tr><tr><td>R-work</td><td>0.1826 (0.2430)</td></tr><tr><td>R-free</td><td>0.2145 (0.2877)</td></tr><tr><td>Number of non-hydrogen atoms</td><td>2308</td></tr><tr><td>macromolecules</td><td>2016</td></tr><tr><td>ligands</td><td>0</td></tr><tr><td>solvent</td><td>292</td></tr><tr><td>Protein residues</td><td>258</td></tr><tr><td>RMS(bonds)</td><td>0.010</td></tr><tr><td>RMS(angles)</td><td>1.04</td></tr><tr><td>Ramachandran favored (%)</td><td>100.00</td></tr><tr><td>Ramachandran allowed (%)</td><td>0.00</td></tr><tr><td>Ramachandran outliers (%)</td><td>0.00</td></tr><tr><td>Rotamer outliers (%)</td><td>0.45</td></tr><tr><td>Clashscore</td><td>1.74</td></tr><tr><td>Average B-factor</td><td>27.34</td></tr><tr><td>macromolecules</td><td>26.40</td></tr><tr><td>solvent</td><td>33.83</td></tr></tbody></table>

Statistics for the highest-resolution shell are shown in parentheses.
<table><caption>Supplemental Table S2. Strains used in this study</caption><thead><tr><td>Strains</td><td>Description and genotype</td><td>Source/References</td></tr></thead><tbody><tr><td>Escherichia coli DH5α</td><td>F-, Δ(argF-lac)U169 phoA supE44 lacZΔM15 recA relA endA thi hsdR gyr</td><td>Laboratory collection</td></tr><tr><td>Escherichia coli BL21(DE3)</td><td>dcm ompT hsdS gal λ DE3</td><td>Laboratory collection</td></tr><tr><td>Escherichia coli W3110</td><td>F-, lambda- IN rrnD-rrnE)1 rph-1</td><td>Laboratory collection</td></tr><tr><td>Saccharomyces cerevisiae W303</td><td>MATa/MATα {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15} [phi<sup>+</sup>]</td><td>Laboratory collection</td></tr></tbody></table>

<table><caption>Supplemental Table S3. Plasmids used in this study</caption><thead><tr><td>Plasmid</td><td>Description</td><td>Source/References</td></tr></thead><tbody><tr><td>pNDM220</td><td>Mini-R1, single-copy vector, LaqI<sup>q</sup>, P<sub>A1/04/03</sub>, Amp<sup>R</sup></td><td>Gotfredsen and Gerdes, 1998</td></tr><tr><td>pBAD33</td><td>Expression vector, AraC, arabinose-inducible, Cm<sup>R</sup></td><td>Guzman et al., 1995</td></tr><tr><td>pNDM220-ARC</td><td><i>P. ananatis</i> LMG 20103 PANA_2924 C-terminal domain cloned into pNDM220</td><td>This study</td></tr><tr><td>pBAD-ARCimm</td><td><i>P. ananatis</i> LMG 20103 PANA_2923 cloned into pBAD33</td><td>This study</td></tr><tr><td>pNDM220-ARC-W273A</td><td>Trp273-to-Ala substitution in pNDM220-ARC</td><td>This study</td></tr><tr><td>pNDM220-ARC-E299A</td><td>Glu299-to-Ala substitution in pNDM220-ARC</td><td>This study</td></tr><tr><td>pNDM220-ARC-W323A</td><td>Trp323-to-Ala substitution in pNDM220-ARC</td><td>This study</td></tr><tr><td>pNDM220-ARC-E356A</td><td>Glu356-to-Ala substitution in pNDM220-ARC</td><td>This study</td></tr><tr><td>pRS416_Gal1</td><td>URA3/Amp, EN6/ARSH4, GAL1 promoter</td><td>Mumberg et al., 1994</td></tr><tr><td>pRS416_Gal1-ARC*</td><td>PANA_2924 C-terminal domain cloned into pRS416_Gal1 and containing a 1 bp (A) deletion at position 435</td><td>This study</td></tr><tr><td>pET-Duet1</td><td>pBR322 ColE1 ori, T7 promoter, Amp<sup>R</sup>, 6×his tag and TEV cleavage site</td><td>Novagen</td></tr><tr><td>pET-hisTEV-ARC-ARCimm</td><td><i>P. ananatis</i> LMG 20103 PANA_2924 C-terminal domain and PANA_2923 cloned into pET-Duet1</td><td>This study</td></tr><tr><td>pET-hisTEV-ARC-E356A</td><td><i>P. ananatis</i> LMG 20103 PANA_2924 C-terminal domain carrying Glu356-to-Ala substitution cloned into pET-Duet1</td><td>This study</td></tr><tr><td>pRSF-Duet1</td><td>RSF1030 (NTP1) ori, T7 promoter, Kan<sup>R</sup></td><td>Novagen</td></tr></tbody></table>
<table><caption>Supplemental Table S4. Primers used in this study</caption><tr><td>pRSF-ARCimm</td><td><i>P. ananatis</i> LMG 20103 PANA_2923 cloned into pRSF</td><td>This study</td></tr><tr><td>pCDF-Duet1</td><td>CloDF13 ori, T7 promoter, Strep<sup>R</sup></td><td>Novagen</td></tr><tr><td>pCDF-ARCimm</td><td><i>P. ananatis</i> LMG 20103 PANA_2923 cloned into pCDF</td><td>This study</td></tr><tr><td>pNDM220-LisARC</td><td><i>L. monocytogenes</i> LS1292 LXG C-terminal domain cloned into pNDM220</td><td>This study</td></tr><tr><td>pBAD-LisARCimm</td><td><i>L. monocytogenes</i> LS1292 immunity against ARC cloned into pBAD33</td><td>This study</td></tr><tr><td>pCH10163</td><td>Plasmid containing <i>E. coli</i> EC93 cdiB and cdiA under their natural promoter</td><td>Willett et al., 2015</td></tr><tr><td>pCH-Cdi::treX-triX</td><td><i>Xenorhabdus bovienii</i> SS-2004 treX-triX cloned into pCH10163</td><td>Dumont et al., 2024</td></tr><tr><td>pCH10163-ARC-ARCimm</td><td><i>P. ananatis</i> LMG 20103 PANA_2924 C-terminal domain and PANA_2923 cloned into pCH10163</td><td>This study</td></tr></table>

<table><thead><tr><td>Primer name</td><td>Sequence 5'- 3'</td><td>Purpose</td></tr></thead><tbody><tr><td>R-pNDM220-seq</td><td>TCTTCGCTATTACGCCAGCT</td><td>Check cloned sequences in pNDM220 vector</td></tr><tr><td>pBAD Fwd</td><td>TCCATAAGATTAGCGGATCC</td><td>Check cloned sequences in pBAD33 vector</td></tr><tr><td>5-pNDM-PANAtox-KpnI</td><td>GCTAGGTACCATGGCAGCC TTTAATGCGTGGCG</td><td>Clone <i>P. ananatis</i> LMG 20103 PANA_2924 C-terminal domain into pNDM220 through KpnI and EcoRI sites</td></tr><tr><td>3-pNDM-PANAtox-EcoRI</td><td>GCTAGAATTCTTAGTTATCC CTTACAAAAATGACTTTTTC CGTCTGTG</td><td>Clone <i>P. ananatis</i> LMG 20103 PANA_2923 into pNDM220 through SalI and HindIII sites</td></tr><tr><td>5-pBAD-PANAimm-Sall</td><td>GCTAGTCGACATGAAAATT ACAGGCACAAGTTCGTATGT GATG</td><td>Clone <i>P. ananatis</i> LMG 20103 PANA_2923 into pNDM220 through SalI and HindIII sites</td></tr><tr><td>3-pBAD-PANAimm-HindIII</td><td>GCTAAAGCTTTTATACAAAT TTCAGGACCATATGAGATCC TTGGG</td><td>Clone <i>P. ananatis</i> LMG 20103 PANA_2924 C-terminal domain into pRS416_Gal1 through KpnI and XbaI sites</td></tr><tr><td>F-paARC-Xba</td><td>CCCCCTCTAGAACTAGTGG ATCCATGGCAGCCTTTAATG CGTGGCGTAA</td><td>Clone <i>P. ananatis</i> LMG 20103 PANA_2924 C-terminal domain into pRS416_Gal1 through KpnI and XbaI sites</td></tr><tr><td>F-pRS416</td><td>CAACATTTTCGGTTTGTATT ACTTC</td><td>Check cloned sequences in pRS416_Gal1 vector</td></tr><tr><td>A-ARC-Pa-W33A</td><td>GCGTCAGGGCGCACTGAAG G</td><td>W273A substitution in <i>P. ananatis</i> LMG 20103 PANA_2924</td></tr><tr><td>B-ARC-Pa-W33A</td><td>GAAAAAGGCACTGTCCGGA TCAG</td><td></td></tr><tr><td>A-ARC-Pa-E59A</td><td>GCATCCACAATCAAAGATA AAAATATTAAAATGCCTG</td><td>E299A substitution in <i>P. ananatis</i> LMG 20103 PANA_2924</td></tr><tr><td>B-ARC-Pa-E59A</td><td>GAGCGTGACACCGCCT</td><td></td></tr></tbody></table>
<table>
  <tr>
    <td>A-ARC-Pa-W83A-2</td>
    <td>CAGAGGATGTCTCCGCGTCT TATG</td>
    <td>W323A substitution in P. ananatis LMG 20103 PANA_2924</td>
  </tr>
  <tr>
    <td>B-ARC-Pa-W83A-2</td>
    <td>CAGCCTTGATGCTCTGCGGG TTATC</td>
    <td></td>
  </tr>
  <tr>
    <td>A-ARC-Pa-E116A</td>
    <td>GCGTTACCGCGCCTGATGGG CAATG</td>
    <td>E356A substitution in P. ananatis LMG 20103 PANA_2924</td>
  </tr>
  <tr>
    <td>B-ARC-Pa-E116A-2</td>
    <td>AACGTTTTCCCATATATTAC CTTCACGCAG</td>
    <td></td>
  </tr>
  <tr>
    <td>F-PaARC-Bmt</td>
    <td>GGGCGCTAGCGCAGCCTTT AATGCGTGGCGTAA</td>
    <td>Clone P. ananatis LMG 20103 PANA_2924 C-terminal domain and PANA_2923 into pET-Duet1-his-TEV through BmtI and HindIII sites</td>
  </tr>
  <tr>
    <td>R-PaImm-Hind</td>
    <td>GACTAAGCTTTTATACAAAT TTCAGGACCATATGAGATCC</td>
    <td></td>
  </tr>
  <tr>
    <td>F-PaImm-Sac</td>
    <td>GGAGGAGCTCATGAAAATT ACAGGCACAAGTTCG</td>
    <td>Clone P. ananatis LMG 20103 PANA_2923 into pRSF-Duet1 and pCDF-Duet1 through SacI and HindIII sites</td>
  </tr>
  <tr>
    <td>ACYCDuetUP1</td>
    <td>GGATCTCGACGCTCTCCCT</td>
    <td>Check cloned sequences in pET-Duet1-his-TEV, pRSF-Duet1, and pCDF-Duet1 vectors</td>
  </tr>
  <tr>
    <td>Duet_down1</td>
    <td>GATTATGCGGCCGTGTACAA</td>
    <td></td>
  </tr>
  <tr>
    <td>5-pNDM-Lis- ARCtox-kpn</td>
    <td>GCTAGGTACCATGGCTAAA GGATTTAATATTGACGAAAT TCATCC</td>
    <td>Clone L. monocytogenes LS1292 LXG C-terminal domain into pNDM220</td>
  </tr>
  <tr>
    <td>3-pNDM-Lis- ARCimm-EcoRI</td>
    <td>GCTAGAATTCTTACTTCCTT TCAAAAATTATTTTTTCAAG TCC</td>
    <td></td>
  </tr>
  <tr>
    <td>F-Lis-ARCimm-sac</td>
    <td>GGAGGAGCTCATGAAAATT ACAGGTAATAGTTCACATGT G</td>
    <td>Clone L. monocytogenes LS1292 immunity against ARC into pBAD33</td>
  </tr>
  <tr>
    <td>R-Lis-ARCimm-xba</td>
    <td>GACTTCTAGATTATTCAAAT GAAATTTGTACTGTATTTTC ATTTGTG</td>
    <td></td>
  </tr>
  <tr>
    <td>F_CDI_plasmid</td>
    <td>CCCAAAGGTTAGACACCAG ACC</td>
    <td>Amplify CDI part from pCH-Cdi::treX-triX</td>
  </tr>
  <tr>
    <td>R_CDI_plasmid</td>
    <td>TGTCGCTGTATTGCGACTCA T</td>
    <td></td>
  </tr>
  <tr>
    <td>F_ARCoperon_CDI _mega</td>
    <td>ATGAGTCGCAATACAGCGA CAGCAGCCTTTAATGCGTGG CGTAAC</td>
    <td>Amplify the PANA_2924 C-terminal domain and PANA_2923 for fusion with CDI components</td>
  </tr>
  <tr>
    <td>R_ARCoperon_CDI _mega</td>
    <td>GGTCTGGTGTCTAACCTTTG GGTTATACAAATTTCAGGAC CATATGAGATCCTTGGG</td>
    <td></td>
  </tr>
  <tr>
    <td>pCH3_F</td>
    <td>CTTCTGATCCTGAAAATCAG GCCATG</td>
    <td>Check cloned sequences in pCH10163-ARC-ARC-imm construct</td>
  </tr>
  <tr>
    <td>pCH3_R</td>
    <td>CCTTGCTACAGATTTCAGAC TCAACTCTC</td>
    <td></td>
  </tr>
</table>
<figure><img src="image_31.png" /></figure>

**Supplemental Figure S1. SEC standard curve and experimental molecular weight determination.**

A standard curve (red dotted line) was established using Bio-Rad molecular weight standards, including bovine thyroglobulin (670 kDa), bovine γ-globulin (158 kDa), chicken ovalbumin (44 kDa), horse myoglobin (17 kDa), and vitamin B₁₂ (1.35 kDa). Because bovine thyroglobin and vitamin B₁₂ fall outside the range of the Superdex 75 column, they were not used to establish the standard curve. The partition coefficient Kₐv was calculated according to the equation Kₐv = (Vₑ - V₀) / (V_c - V₀), where Vₑ is the elution volume of the analyte, V_c is the column bed volume (determined from vitamin B₁₂ elution), and V₀ is the void volume (determined from thyroglobulin elution). The linear regression equation and corresponding R² value are shown in the plot. MW, molecular weight.
<figure><img src="image_32.png" /><figcaption>**Supplemental Figure S2.** Comparison of the crystal structures of the two chains of the PANA_2924 C-terminal extension asymmetric unit. Superimposition of the crystal structures of chains A (blue) and B (red) of the PANA_2924 C-terminal domain. The dotted lines highlight the positions of the loops that are missing in one structure but visible in the other.</figcaption></figure>
A

W273
PANA_2924 KSLINLKEIEP--QLATDP---DSAFFWSGRTEGVGGPDVAAEAIAK-SRG
...:..:| :|.|..  ...| |:| :|:... :|.
CD38 ----TEEDYQPLMKLGTQTVCNKILLWS-RIK-----DLAHQFTQVQRD
W125

E299
PANA_2924 GVTLESTI------------------KDKNIKMPEWDFD---NPQSIKAWE
..|..:|: |......|:|..| |:|:|:.
CD38 MFTLEDTLLGYLADDLTWCGEFDTSKINYQSCPDWRKDCSNNPVSV-FWK
E146
W189

E356
PANA_2924 DVSASYAKQVSGEVRAVVGQS----LREGNIWENVELPRLMGNDNVTKIT
..|..:|:.....|..::..|  ..:..:|:|..|...
CD38 TVSRRFAEAACDVVHVMLDGSRSKIFDKDSTFGSVEVHNLQPE-----KVQ
E226

PANA_2924 TI------------------DP-LSQTEKVIFVRDN--------
|:| |:..:|:|..|:|.
CD38 TLEAWVIHGGRESDRDLCQDPTIKELESIISKRNIQFSCKNIY

B

<figure><img src="image_33.png" /><figcaption>2924-C (PDB: 9RO8)
ARCtox</figcaption></figure>

<figure><img src="image_34.png" /><figcaption>CD38 (PDB: 2PGJ)</figcaption></figure>

Supplemental Figure S3. Comparison of the amino acid sequences and crystal structures of the PANA_2924 C-terminal extension (PDB: 9RO8) and human CD38 (PDB: 2PGJ). (A) Sequence alignment of PANA_2924 and CD38 C-terminal domains, highlighting catalytic residues. PANA_2924 and CD38 catalytic residues are shown in blue and red, respectively. The alignment was generated using the EMBOSS Needle pairwise alignment tool. (B) Structural arrangement of conserved catalytic residues (shown in grey) within the NAD⁺/NADP⁺ binding pockets of the PANA_2924 C-terminal extension (left) and CD38 (right).
A

<figure><img src="image_35.png" /></figure>

B

<figure><img src="image_36.png" /><figcaption>**Supplemental Figure S4.** Confidence scores of the AlphaFold3 structural model of the ARC^tox/ARC^imm complex. (A) Structural model of the ARC^tox-ARC^imm complex colored by predicted Local Distance Difference Test (pLDDT) scores, ranging from blue (high confidence, >90) to orange (low confidence, <50). The green circle indicates ARC^imm. (B) Predicted Aligned Error (PAE) plot showing the expected positional error (in Å) for each residue pair, with dark green indicating high confidence and lighter green indicating lower confidence.</figcaption></figure>
<figure><img src="image_37.png" /></figure>

<figure><img src="image_38.png" /></figure>

<figure><img src="image_39.png" /></figure>

<figure><img src="image_40.png" /></figure>

**Supplemental Figure S5. Control chromatograms.** Mixed-mode HPLC chromatograms of the products of the reactions of NAD⁺ (A) and NADP⁺ (B) in the presence of buffer (20 mM ammonium formate, pH2.9, for NAD⁺ or 10 mM ammonium formate, pH3, for NADP⁺), as well as for the ARC<sup>tox</sup> alone in the presence of ammonium formate buffers (C and D). Note that a weak peak corresponding to nicotinamide is always visible for NADP⁺ alone.
(2)

<figure><img src="image_41.png" /></figure>

RMSD = 0.649

(3)

<figure><img src="image_42.png" /></figure>

RMSD = 0.617

(4)

<figure><img src="image_43.png" /></figure>

RMSD = 0.395

(5)

<figure><img src="image_44.png" /></figure>

RMSD = 0.668

(6)

<figure><img src="image_45.png" /></figure>

RMSD = 0.394

(7)

<figure><img src="image_46.png" /></figure>

RMSD = 0.732

(8)

<figure><img src="image_47.png" /></figure>

RMSD = 0.761

(9)

<figure><img src="image_48.png" /></figure>

RMSD = 0.552

(10)

<figure><img src="image_49.png" /></figure>

RMSD = 0.47

(11)

<figure><img src="image_50.png" /></figure>

RMSD = 0.487

(12)

<figure><img src="image_51.png" /></figure>

RMSD = 0.888

Supplemental Figure S6. Alignment of the *P. ananatis* ARC<sup>tox</sup> crystal structure (red) with AlphaFold3 structural models of ARC domains. 2, *Agrobacterium rubi* NTF28035; 3, *Burkholderia gladioli* WP_186165364; 4, *Listeria monocytogenes* WP_120135998; 5, *Gordonia jinhuaensis* WP_188589035; 6, *Lactiplantibacillus plantarum* WP_076633504; 7, *Stenotrophomonas maltophilia* OCK46403; 8, *Nonomuraea fuscirosea* WP_364663299; 9, *Marinobacter shengliensis* WP_138437386; 10, *Pendulispora albinea* WP_394825747; 11, *Allomuricauda sp.* RPG31737; 12, *Streptomyces mirabilis* WP_388533272. Root-Mean-Square Deviation (RMSD) values are indicated below each superimposition. The pLDDT scores for AlphaFold3 structural model for each ARC domain are shown in Supplemental Figure S7.
<figure><img src="image_52.png" /><figcaption>**Supplemental Figure S7. AlphaFold3 structural models of ARC domains** colored by pLDDT scores, ranging from blue (high confidence, >90) to orange (low confidence, <50). 2, *Agrobacterium rubi* NTF28035; 3, *Burkholderia gladioli* WP_186165364; 4, *Listeria monocytogenes* WP_120135998; 5, *Gordonia jinhuaensis* WP_188589035; 6, *Lactiplantibacillus plantarum* WP_076633504; 7, *Stenotrophomonas maltophilia* OCK46403; 8, *Nonomuraea fuscirosea* WP_364663299; 9, *Marinobacter shengliensis* WP_138437386; 10, *Pendulispora albinea* WP_394825747; 11, *Allomuricauda* sp. RPG31737; 12, *Streptomyces mirabilis* WP_388533272.</figcaption></figure>