# Toxin \(\zeta\) Reduces the ATP and Modulates the Uridine Diphosphate-N-acetylglucosamine Pool
## 2 Results
### Toxin \(\zeta\) Is a UNAG-Dependent ATPase
When [\(\gamma\)32P]-ATP was replaced by [\(\alpha\)32P]-ATP, only the accumulation of the [\(\alpha\)32P]-ADP/ADP product was observed (Figure 2A, lanes 7-14). To test whether the UNAG-P or the ADP formed during the reaction interfere with \(\zeta\)-mediated ATP hydrolysis, stoichiometric amounts of UNAG and [\(\alpha\)32P]-ATP/ATP (2 mM) were incubated with \(\zeta\) (500 nM) for 30 min. Toxin \(\zeta\) hydrolyzed \(>\) 95% of the ATP substrate to convert it into [\(\alpha\)32P]-ADP/ADP and P\({}_{\rm i}\) (Figure 2B). In a second step, fresh [\(\alpha\)32P]-ATP/ATP (+2 mM) in the presence or the absence of an ATP regeneration system (ATP-Reg) was added to the previous reaction, and samples were further incubated for 30 min. About 80% of the newly added [\(\alpha\)32P]-ATP/ATP was converted to product [\(\alpha\)32P]-ADP/ADP and P\({}_{\rm i}\) (Figure 2B, dark grey bar). Similar results were observed when the ATP-Reg was added (Figure 2B, empty bar). In the absence of UNAG, toxin \(\zeta\) did not hydrolyze ATP, even in the presence of the lactate dehydrogenase/pyruvate kinase ATP-Reg system (data not shown). Unlike the distantly related AvrRxo1 toxin [42], toxin \(\zeta\) did not transfer the ATP-P\(\gamma\) to the NAD formed during ATP-regeneration. In the presence of the ATP-Reg system, the maximal rate of \(\zeta\)-mediated ATP hydrolysis was maintained for the first 8 h, and slightly  
Figure 2: Toxin \(\zeta\) hydrolyzes ATP in presence of UNAG. (**A**) Toxin \(\zeta\) (500 nM), 2 mM of ATP (with fixed concentration of [\(\alpha\)32P]-ATP or [\(\gamma\)32P]-ATP, at a 1:100,000 ratio) and decreasing concentration of UNAG were incubated for 60 min at 30 \({}^{\circ}\)C in buffer D. ATP hydrolysis was analyzed by TLC performed on PEI cellulose plates in buffer E as the mobile phase. As control in lanes 6 and 15 UNAG was omitted. (**B**) Schematic representation of the reaction. Toxin \(\zeta\) (500 nM) was incubated with UNAG (2 mM) and ATP (2 mM, with a fixed concentration of [\(\alpha\)32P]-ATP, 10 nM) for 30 min in buffer D. Then, the reaction was divided in three parts. One aliquot was loaded onto a TLC. To the second and third aliquots, ATP or ATP and a regeneration system was added, and the reaction incubated for 60 min at 30 \({}^{\circ}\)C in buffer D. The graph represents the percentage of remaining ATP in the different conditions. The reaction without \(\zeta\) was used as control. The results are expressed as the mean \(\pm\) SEM of \(>\)3 independent experiments. The + and - symbols denote presence or the absence of the indicated compound. reduced after 72 h of incubation (data not shown). It is likely that: (i) toxin \(\zeta\) is a UNAG-dependent ATPase that phosphorylates UNAG with low efficiency (up to 7% of total UNAG); (ii) the accumulation of up to 20-fold excess of ADP does not inhibit the activity of toxin \(\zeta\); and (iii) traces of UNAG-P marginally reduce the activity of toxin \(\zeta\). This is consistent with the affinity binding predictions that revealed relatively lower affinities for ADP and UNAG than for ATP and UNAG-P (Table 1).