Role of the cysteine residues in Arabidopsis thaliana cyclophilin CYP20-3 in peptidyl-prolyl cis-trans isomerase and redox-related functions.
Cyps (cyclophilins) are ubiquitous proteins of the immunophilin superfamily with proposed functions in protein folding, protein degradation, stress response and signal transduction. Conserved cysteine residues further suggest a role in redox regulation. In order to get insight into the conformational change mechanism and functional properties of the chloroplast-located CYP20-3, site-directed mutagenized cysteine-->serine variants were generated and analysed for enzymatic and conformational properties under reducing and oxidizing conditions. Compared with the wild-type form, elimination of three out of the four cysteine residues decreased the catalytic efficiency of PPI (peptidyl-prolyl cis-trans isomerase) activity of the reduced CYP20-3, indicating a regulatory role of dithiol-disulfide transitions in protein function. Oxidation was accompanied by conformational changes with a predominant role in the structural rearrangement of the disulfide bridge formed between Cys(54) and Cys(171). The rather negative E(m) (midpoint redox potential) of -319 mV places CYP20-3 into the redox hierarchy of the chloroplast, suggesting the activation of CYP20-3 in the light under conditions of limited acceptor availability for photosynthesis as realized under environmental stress. Chloroplast Prx (peroxiredoxins) were identified as interacting partners of CYP20-3 in a DNA-protection assay. A catalytic role in the reduction of 2-Cys PrxA and 2-Cys PrxB was assigned to Cys(129) and Cys(171). In addition, it was shown that the isomerization and disulfide-reduction activities are two independent functions of CYP20-3 that both are regulated by the redox state of its active centre.
Laxa
Miriam
M
Biochemistry and Physiology of Plants, Faculty of Biology, W5, Bielefeld University, 33501 Bielefeld, Germany.
König
Janine
J
Dietz
Karl-Josef
KJ
Kandlbinder
Andrea
A
D017360:Arabidopsis / Q000201:enzymology* / Q000254:growth & development; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D021983:Cyclophilins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine; D014644:Genetic Variation; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D018749:RNA, Plant / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D029681:Arabidopsis Proteins; D018749:RNA, Plant; D011994:Recombinant Proteins; D021983:Cyclophilins; C000616093:cyclophilin 20-3, Arabidopsis; D003545:Cysteine