# Toxin \(\zeta\) Reduces the ATP and Modulates the Uridine Diphosphate-N-acetylglucosamine Pool
## 4 Materials and Methods
### Biochemical Assays  
_Streptococcus pyogenes_ wt toxin \(\zeta\) and its mutant variants were over-expressed in _E. coli_ BL21(DE3) cells in the presence of rifampicin and purified. Toxin \(\zeta\), free of the \(\varepsilon_{2}\) antitoxin, was purified as reported [24]. Toxin \(\zeta\) variants \(\zeta\)D67A, \(\zeta\)E100A, \(\zeta\)E116A, \(\zeta\)T128A, \(\zeta\)R158A and \(\zeta\)R171A were purified in two steps using a similar protocol [24]. In short, cells were broken using a French Press in buffer A (50 mM phosphate buffer pH 7.5, 100 mM NaCl, 5% glycerol). The soluble toxin variants were bound to a Ni-NTA column, and eluted using an imidazole gradient (2 to 75 mM). In a second step, protein containing fractions were diluted to 25 mM NaCl and passed through a Q Sepharose column. Proteins were eluted in buffer B (50 mM Tris HCl pH7.5) containing a gradient of NaCl (25 to 150 mM). The fractions containing \(\zeta\), \(\zeta\)D67A, \(\zeta\)E100A, \(\zeta\)E116A, \(\zeta\)T128A, \(\zeta\)R158A and \(\zeta\)R171A were dialyzed against buffer C (50 mM Tris HCl pH7.5, 100 mM NaCl) containing 50% glycerol and stored at \(-20\)\({}^{\circ}\)C.  
Thin layer chromatography (TLC) assays were used to measure the ATPase activity of the purified toxin \(\zeta\). The toxin was incubated using either fixed or increasing UNAG concentrations in buffer D (50 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mM MgCl\({}_{2}\)) at 30 \({}^{\circ}\)C. The toxin \(\zeta\), concentration chosen for the TLC assays was the one that degraded 95% of the ATP substrate in 60 min at 30 \({}^{\circ}\)C. The reaction was started adding radiolabeled 2 mM ATP ([\(\alpha\)\({}^{32}\)P]-ATP/ATP or [\(\gamma\)\({}^{32}\)P]-ATP/ATP at a 1:100,000 ratio), and stopped after 60 min by adding 25 mM EDTA. From each reaction 2-5 uL were spotted in 20 \(\times\) 20 cm TLC PEI cellulose plates, and chromatography was performed for 120 min in a TLC chamber containing running buffer E [0.85 M KH\({}_{2}\)PO\({}_{4}\) (pH 3.4)] as eluent. Dried TLC plates were analyzed with a PMI molecular imager (BioRad, Hercules, CA, USA).  
The ATPase activity of wt toxin \(\zeta\) and its variants (\(\zeta\)D67A, \(\zeta\)E100A, \(\zeta\)E116A, \(\zeta\)T128A, \(\zeta\)R158A and \(\zeta\)R171A) was also measured using an ATP/NADH coupled assay [62; 63]. The toxin was incubated with increasing ATP concentrations in the presence of a fixed UNAG (10 mM) or fixed ATP (10 mM) and variable of UNAG for the indicated times, in buffer D (50 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mM MgCl\({}_{2}\)). The reactions additionally contained the NADH enzyme mix (300 uM NADH, 100 U/mL of lactate dehydrogenase, 500 U/mL pyruvate kinase, and 2.5 mM phosphoenolpyruvate) in buffer D and had a volume of 50 uL. The ATPase activity was determined monitoring the disappearance of absorbance at 340 nm, due to NADH conversion to NAD, using a Shimadzu CPS-20A dual-beam spectrophotometer. A standard curve with known amounts of NADH was obtained and used to convert the drop-in absorbance/time to ADP concentration/time [63]. V\({}_{\text{max}}\) and K\({}_{\text{m}}\) values were calculated by constructing Michaelis-Menten plots using R and the Graphpad Prism Software (6.0).