Role of the cysteine residues in Arabidopsis thaliana cyclophilin CYP20-3 in peptidyl-prolyl cis-trans isomerase and redox-related functions

• Published in: Biochemical journal 2007-01, Vol.401 (1), p.287-297
• Main Authors: Laxa, Miriam, König, Janine, Dietz, Karl-Josef, Kandlbinder, Andrea
• Format: Article
• Language: English
• Subjects: Arabidopsis - enzymology; Arabidopsis - growth & development; Arabidopsis Proteins - chemistry; Arabidopsis Proteins - genetics; Arabidopsis Proteins - metabolism; Cyclophilins - chemistry; Cyclophilins - genetics; Cyclophilins - metabolism; Cysteine; Genetic Variation; Kinetics; Mutagenesis, Site-Directed; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; RNA, Plant - genetics; RNA, Plant - isolation & purification
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/BJ20061092

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.