# Toxin \(\zeta\) Reduces the ATP and Modulates the Uridine Diphosphate-N-acetylglucosamine Pool
## 2 Results
### Toxin \(\zeta\) Is a UNAG-Dependent ATPase  
To test whether the hydrolysis of ATP by toxin \(\zeta\) is more efficient than the phosphotransfer of P\({}_{\rm i}\) to UNAG, the protein was incubated with its potential substrates and the products were analyzed. In the absence of UNAG, toxin \(\zeta\) (500 nM) did not hydrolyze ATP (Figure 2A, lanes 6 and 15) [24; 25]. In the presence of 1 mM UNAG, toxin \(\zeta\) (500 nM), at half of its physiological concentration [24], hydrolyzed ~95% of the ATP substrate in a 60 min reaction (Figure 2A, lanes 3 and 10). In the presence of limited \(\zeta\) (60 nM), the reaction reached a steady state rate of ATP hydrolysis near the previously observed K\({}_{\rm cat}\) of ~1350 \(\pm\) 165 min\({}^{-1}\) (Supplementary Figure S1A,B). Previously, it has been shown that incubation of toxin \(\zeta\) with its cognate \(\epsilon_{2}\) antitoxin for 5 min at \(\zeta\):\(\epsilon_{2}\) ratios of 1:1, inhibits \(\zeta\)-mediated ATP hydrolysis in the presence of saturating UNAG concentrations, suggesting that the \(\epsilon_{2}\) antitoxin is necessary and sufficient to inactivate toxin \(\zeta\)[25]. A distant member of the \(\zeta\)/PezT toxin superfamily (toxin AvrRxo1) has been proposed to transfer the ATP-Py to single-stranded (ss) or double-stranded (ds) DNA [42]. When UNAG was replaced by ssDNA or dsDNA, toxin \(\zeta\) did not catalyze [\(\gamma\)-\({}^{32}\)P]-ATP/ATP hydrolysis (Figure S1B), suggesting that ssDNA or dsDNA are not the target of the \(\zeta\) toxin. Indeed, no radiolabeled linear ssDNA or dsDNA was detected. In the presence of increasing UNAG and fixed ATP concentrations, purified toxin \(\zeta\) (500 nM) hydrolyzed [\(\gamma\)\({}^{32}\)P]-ATP/ATP producing \({}^{32}\)P\({}_{\rm i}\)/P\({}_{\rm i}\) and ADP, with \({}^{32}\)P\({}_{\rm i}\)/P\({}_{\rm i}\) co-migrating with the front in our experimental conditions (Figure 2A, lanes 1-5). More than 95% of the 2 mM [\(\gamma\)\({}^{32}\)P]-ATP/ATP was converted to product in 60 min (Figure 2A, lane 1). If the Py phosphotransfer reaction was coupled to ATP hydrolysis, a large fraction of UNAG should be converted to UNAG-P. Mass spectrometry analyses confirmed that > 95% of the ATP (molecular mass 507.18 Da, observed as peaks of 506.06 and 628.13 peaks) was hydrolyzed, as judged by the reduction of ATP and its Na-bound variant, and by the increment of the ADP peak (Figure S2A,B). Concomitantly, the UNAG peak (molecular mass 607.35 Da, observed as peaks of 606.15 and 628.13 Da) was slightly reduced and small peaks corresponding to UNAG-P (molecular mass 687.35 Da, observed as 686.12 and 708.10 Da) were detected (Figure S2B), when compared with the mock reaction (no \(\zeta\) added) (Figure S2A). Similar results have been reported previously [24; 25]. It is likely that even with an ~8-fold increase in toxin \(\zeta\) concentration UNAG phosphorylation was limited, and >20 ATP molecules were hydrolyzed for each P\(\gamma\) transferred to UNAG. Toxin \(\zeta\) is specific for UNAG, because its ATPase activity in the presence of other UDP-activated sugars (e.g., UDP-glucose and UDP-N-acetylgalactosamine) is reduced >50-fold when compared to UNAG [23; 25]. \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Condition** & **ATP** & **ADP** & **UNAG** & **UNAG-3P** \\ \hline H-bonds & 4 & 1 & 5 & 8 \\ Contacts & 12 & 7 & 14 & 16 \\ Atom contacts & 92 & 35 & 60 & 82 \\ DSX & \(-\)110.835 & \(-\)61.036 & \(-\)99.592 & \(-\)119.225 \\ Xscore & \(-\)6.73 & 4.57 & \(-\)7.36 & \(-\)7.81 \\ \hline \hline \end{tabular} H-bonds: total number of H-bonds between UNAG and \(\zeta\). Contacts: number of amino acids making contacts with substrate and products. Atom contacts: total number of contact atoms. DSX: DrugScore DSX score and Xscore: estimated binding energy in Kcal/mol (the lower scores indicate stronger binding affinity).  
\end{table} 
Table 1: Predicted interaction between \(\zeta\) and its ligands.