ARS | Publication request: Site-Directed Mutagenesis of Negatively Charged Residues in the Carboxyl Terminus of the 46-Kda Isoform of Arabidopsis Rubisco Activase

Submitted to: American Society of Plant Biologists Annual Meeting
Publication Type: Abstract
Publication Acceptance Date: March 15, 2004
Publication Date: July 26, 2004
Citation: Wang, D., Portis Jr, A.R. 2004. Site-directed mutagenesis of negatively charged residues in the carboxyl terminus of the 46-kDa isoform of Arabidopsis Rubisco activase [abstract]. American Society of Plant Biologists Annual Meeting. Available: http://abstracts.aspb.org/pb2004/public/P40/7654.html.

Technical Abstract: Two cysteine residues, C392 and C411, in the carboxyl terminus of the 46-kDa isoform of Rubisco activase are required to maintain the greater ADP sensitivity and form a disulfide bond that is reduced by thioredoxin-f. There are six negatively charged residues (E390, D394, E398, D401, D407 and D408) located near these two Cys residues. An electrostatic interaction between them and positively charged residues located in the ATP binding site has been suggested to stabilize the conformation of the carboxyl terminus after oxidation (Photosyn. Res. 68: 29-37, 2001). To further explore this hypothesis, E390, D394, E398, D401, D407, D408 were changed to Ala by site-directed mutagenesis. Two double mutants D394A/E398A and E390A/D401A were also made. Analysis of the purified recombinant proteins showed that D394, E398, D407, D394A/E398A and E390A/D401A were less sensitive to ADP inhibition than the wild type 46-kDa isoform. Those mutant enzymes (except D407A) also exhibited higher Rubisco activation activities than wild type enzyme. To examine the possibility that mutation might affect the thioredoxin-mediated redox regulation of the 46-kDa isoform, the equilibrium redox midpoint potentials for the mutant D394A, E398A and wild type enzymes were compared. Slight differences in midpoint potentials were observed. These results are consistent with the hypothesis that the carboxyl terminal domain is important in the redox regulation. To further explore the structural stability of carboxyl terminal domain, partial digestions with appropriate proteases will be applied under the reduced and oxidized conditions. HPLC and MS analysis will be used for the identification of conformation changes in carboxyl terminal region.