Studies with R570A strain resulted in 60% reduction in toxicity after 8 h postinduction as shown in Fig. 2c, which indicate the importance of this residue in the activity Torin 1 of catalytic domain. Although in primary sequence, R570 is located far from H535, H538 and E542, due to the protein conformation, it became a part of the cleft formed by these amino acids as shown in Fig. 2b. Moreover, it might be possible that
positive charge on the R570 assists in the binding of RNA at putative active site by neutralizing the negative charges present on the backbone of RNA due to phosphate group. Interestingly, there was no reduction in toxicity in K564A strain whose growth profile was similar to wild type as shown in Fig. 2c. In three-dimensional structure of catalytic domain as shown in Fig. 2a, K564 lies very far from other conserved residue hence it is not part of putative active site but may assist in binding of RNA to the active due to its positive charge. Hence, we concluded that D535 and H538
act as acid–base pair to hydrolyse RNA, and D535, H538, E542 and R570 formed the active site in catalytic PD-0332991 manufacturer domain of xenocin. To confirm that the loss of endogenous toxicity in catalytic domain variant strains was not due to the conformational change of the protein induced by site-directed mutagenesis, site-directed mutations were performed in pJC4 construct containing catalytic-immunity domain complex at all the six conserved sites. Wild-type catalytic-immunity domain complex and all the mutant complexes were purified with Ni-NTA chromatography under native conditions. Further, domains were separated and purified by ion exchange chromatography as discussed in ‘Material and methods’. The homogeneity of purified catalytic Etofibrate domain variants was further confirmed by Western blot analysis using anti-rabbit serum generated against full-length xenocin protein as shown in Fig. 3a. Expression and purification of the immunity domain with the mutated catalytic domains indicate that mutation did not affect the formation of stable protein complexes. From this observation,
we may hypothesize that catalytic domain consists of two functional regions. N′ terminal region of catalytic domain is responsible for the binding of immunity protein, whereas C′ terminal consists of active site. To validate the endogenous toxicity assay, in vitro RNase degradation assay was performed with recombinant catalytic wild-type domain and its mutant variants. Result showed that total RNA isolated from E. coli BL 21(DE3)/pLysS cell was intact and not degraded when incubated with purified recombinant domain D535A and H538A mutant protein as shown in Fig. 3b lane 2 and 3, respectively. Moreover, these results were comparable to negative control experiment, which was performed without protein as shown in Fig. 3b lane 1. Therefore, we inferred that the D535 and H538 are the key amino acid residues of the active site of the catalytic domain of xenocin.