Title:Role of Cysteine Residues in Regulation of Peptidyl-prolyl cis-trans Isomerase Activity of Wheat Cyclophilin TaCYPA-1
Volume: 24
Issue: 6
Author(s): Gundeep Kaur, Harpreet Singh, Kirandeep Kaur, Suchismita Roy, Ashwani Pareek and Prabhjeet Singh*
Affiliation:
- Department of Biotechnology, Guru Nanak Dev University, Amritsar-143005,India
Keywords:
Cyclophilin, cysteine, redox regulation, peptidyl-prolyl cis-trans isomerase, wheat, heat stress.
Abstract: 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 Cu
2+ 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 (K
cat/K
m) of TaCYPA-1
C40S (0.37 X 10
6 M
-1 s
-1) and TaCYPA-
1
C122S (0.31 X 106 M-1 s-1) were significantly lower as compared to the native TaCYPA-1 (1.33 X
10
6 M
-1 s
-1), whereas K
cat/K
m of the double mutant TaCYPA-1
C40S/C122S was significantly higher
(2.36 X 10
6 M
-1 s
-1). Compared to the wild-type TaCYPA-1, the different mutants also showed differential
sensitivity to Cu
2+. Furthermore, the results of this study also revealed that despite lacking
PPIase activity, the mutant TaCYPA-1
C126P 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-1
C126P, 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.
