Properties and mutation studies of a bacteriophage-derived chimeric recombinant staphylolytic protein P128: Comparison to recombinant lysostaphin.

Abstract

P128 is a chimeric anti-staphylococcal protein having a catalytic domain from a Staphylococcus bacteriophage K tail associated structural protein and a cell wall targeting domain from the Staphylococcus bacteriocin-lysostaphin. In this study, we disclose additional properties of P128 and compared the same with lysostaphin. While lysostaphin was found to get inactivated by heat and was inactive on its parent strain S. simulans biovar staphylolyticus, P128 was thermostable and was lytic towards S. simulans biovar staphylolyticus demonstrating a difference in their mechanism of action. Selected mutation studies of the catalytic domain of P128 showed that arginine and cysteine, at 40th and 76th positions respectively, are critical for the staphylolytic activity of P128, although these amino acids are not conserved residues. In comparison to native P128, only the R40S mutant (P301) was catalytically active on zymogram gel and had a similar secondary structure, as assessed by circular dichroism analysis and in silico modeling with similar cell binding properties. Mutation of the arginine residue at 40th position of the P128 molecule caused dramatic reduction in the V_max (∆OD₆₀₀ [mg/min]) value (nearly 270 fold) and the recombinant lysostaphin also showed lesser V_max value (nearly 1.5 fold) in comparison to the unmodified P128 protein. The kinetic parameters such as apparent K_m (K_m^APP) and apparent K_cat (K_cat^APP) of the native P128 protein also showed significant differences in comparison to the values observed for P301 and lysostaphin.