# Toxin \(\zeta\) Reduces the ATP and Modulates the Uridine Diphosphate-N-acetylglucosamine Pool
## 2 Results
### Alanine Mutagenesis of Relevant Residues  
From the structure of the \(\zeta\epsilon_{2}\zeta\), the UNAG-\(\zeta\epsilon_{2}\zeta\)-UNAG complexes [19; 22; 23], the known structures of phosphotransferases in the ATP bound form [19; 22] and the analysis of the interaction between wild type (wt) toxin \(\zeta\) and ATP, we predicted that residues K46, D67, E100, E116, T128, R158 and R171 might play crucial roles in substrate binding. We performed an in silico alanine scanning of the \(\zeta\epsilon_{2}\zeta\) complex and of toxin \(\zeta\) to understand the effect of mutations in the K46, D67, E100, E116, T128, R158 and R171 residues on UNAG binding using TRITON [43] and Modeller v9 [44]. In the toxin \(\zeta\)-UNAG structure, residues D67, E100, E116 and T128 were in direct contact with UNAG. Residues R158 and R171, which form hydrogen bonds with the phosphate groups, might interact with Mg\({}^{2+}\) and stabilize the negative charge of ATP, while K46 should interact with the \(\beta\) and \(\gamma\) phosphates of ATP (Figure 3B). The role of the \(\zeta\)K46A mutant variant in ATP binding has been reported previously [22]. Figure 3: Toxin \(\zeta\) residues D67, E116, R158 and R171 are relevant for enzyme activity in vivo. **(A)** Potential \(\zeta\) residues relevant for UNAG binding. In blue the mutated residues that result in elimination of the toxicity of the protein; **(B)** In vivo assays for toxicity. In the absence of the antitoxin \(\epsilon\) gene, plasmid DNA (200 ng) bearing wt \(\zeta\) or its mutant variants (R171A, R158A, T128A, E116A, E100A, and D67A) were transformed into _E. coli_ BL21 (DE3) [pLysS] competent cells and appropriated dilutions were platted on LB agar plates containing 500 \(\upmu\)M IPTG. The relative transformation frequency, indicated as the mean \(\pm\) SEM of > 3 independent experiments, of wt or each mutant with respect to a transformation control with plasmid-borne \(\zeta\) and \(\epsilon\) genes (100%) was represented. All the mutants showed a disruption of the H-bonding and contact patterns, with absence of contacts between UNAG and the mutated residue (Table 2). The effect of some of the mutants might be smaller and due to roles unrelated to UNAG binding according to affinity predictions. From the data presented in Table 2, it is likely that the role of D67 is catalytic, E116 may coordinate Mg\({}^{2+}\), R158 may bind ATP, and that E100 and T128 likely have a small indirect effect through structural changes (Table 2). Residue E100 contributes to stabilize UNAG-O2' via electrostatic and H-bonding interactions. When E100 is substituted for an alanine residue, UNAG-O2' H-bonds with N537 instead, losing the H-bonds with R129, T118 and D67, which no longer contribute to locate UNAG in place for the transfer reaction. These changes suggest that UNAG binding and phosphorylation might be hampered in \(\zeta\)E100A, facilitating an increase of ATP hydrolysis. The predicted binding scores of suitably oriented conformers is potentially relevant, as it may affect the efficiency of the enzyme by limiting the reaction rate through the association/dissociation rates of substrates and/or products (Table 2). To corroborate these hypotheses experimentally, the toxin \(\zeta\) mutant variants were constructed (Supplementary Table S1). The plasmid-bearing wt toxin \(\zeta\), as well as its variants lacking or containing the \(\varepsilon\) antitoxin gene, were purified, and then used to transform plasmid-free cells. DNA from the plasmid-borne \(\varepsilon\) and \(\zeta\) genes (pCB920) transformed _E. coli_ cells at a frequency arbitrarily fixed as 100% (\(\zeta+\varepsilon_{2}\) condition), and with similar efficiency to the empty vector DNA (Figure 3B). In the absence of the \(\varepsilon\) antitoxin gene, the plasmid-borne wt \(\zeta\) (pCB1024) and \(\zeta\)E100A (pCB1029) did not transform _E. coli_ competent cells (Figure 3B), suggesting that wt, \(\zeta\)E100A and to a lesser extent the \(\zeta\)T128A variant were active and toxic.