Background: The wheat cyclophilin, TaCYPA-1, shows peptidyl-prolyl cis-trans isomerase (PPIase) activity. However, the significance of different cysteine residues in regulation of PPIase activity of this protein has not been investigated.Objectives: The main objective of this study was to analyze the role of different disulphide linkages in redox mechanisms that modulate the PPIase activity of TaCYPA-1. Method: Site-directed mutants of TaCYPA-1 were generated by substituting cysteine residues at positions -40 and -122 with serine and -126 with proline. The recombinant proteins were expressed in Escherichia coli and purified. The effect of Cu2+ and N-ethylmaleimide was studied on PPIase activity of the purified recombinant cyclophilins for analyzing the role of different cysteine residues in the modulation of enzyme activity. The changes in secondary structure of TaCYPA-1 and its mutants were analysed by recording far UV CD spectra. The effect of different cysteine substitutions on thermotolerance of E. coli was studied by monitoring the cell growth at 47 °C after 2 h, 3 h and 5 h of heat stress. Results: The catalytic efficiencies (Kcat/Km) of TaCYPA-1C40S (0.37 X 106 M-1 s-1) and TaCYPA- 1C122S (0.31 X 106 M-1 s-1) were significantly lower as compared to the native TaCYPA-1 (1.33 X 106 M-1 s-1), whereas Kcat/Km of the double mutant TaCYPA-1C40S/C122S was significantly higher (2.36 X 106 M-1 s-1). Compared to the wild-type TaCYPA-1, the different mutants also showed differential sensitivity to Cu2+. Furthermore, the results of this study also revealed that despite lacking PPIase activity, the mutant TaCYPA-1C126P was able to confer partial protection against heat stress. Conclusion: This study revealed that mutation of different cysteine residues in TaCYPA-1 results in differential effect on PPIase activity. It was also observed that of the two pairs of cysteine residues i.e. Cys40/Cys168 and Cys122/Cys126, the latter appears to play major role in redox regulation of TaCYPA-1. The mutant TaCYPA-1C126P, though lacking PPIase activity, was able to impart partial tolerance to heat stress in E. coli, suggesting other functions besides cis to trans isomerisation. This study, therefore, provides new insights into the regulation of plant cyclophilins.
Keywords: Cyclophilin, cysteine, redox regulation, peptidyl-prolyl cis-trans isomerase, wheat, heat stress.