Role of D 535 and H 538 in endogenous toxicity of xenocin from X enorhabdus nematophila

Bacteriocins are the toxic proteins produced by bacteria under stress condition to inhibit the growth of closely related bacterial strain(s). In our earlier study, purified recombinant xenocin–immunity protein complex from X enorhabdus nematophila showed detrimental effect on six different insect gut residing bacteria. In this study, endogenous toxicity assay with xcinA and its catalytic domain under tightly regulated ara promoter was performed. Multiple sequence alignment and homology modelling revealed six conserved amino acid residues in the catalytic domain of xenocin. Site‐directed mutagenesis was performed in all the conserved residues, followed growth profile analysis of all the mutants by endogenous toxicity assay. Among the six different conserved sites in catalytic domain of xenocin, we have identified one position where mutation resulted in no measurable reduction in the endogenous toxicity ( K 564), three positions with measurable reduction in the endogenous toxicity ( E 542, H 551 and R 570) and two positions where mutation caused a significant reduction in the toxicity ( D 535 and H 538). Endogenous toxicity assay is validated by in vitro RNA degradation assay. Structural integrity of purified recombinant proteins was confirmed through circular dichroism and fluorescence spectroscopy. Our results indicate that D 535 and H 538 act as the acid–base pair for RNA hydrolysis.